KR20220011813A - High Temperature Resistant Transformed Plant - Google Patents

Abstract

The present invention relates to a method for imparting resistance to high temperature stress, characterized in that the nucleic acid encoding the protein of the following (a) or (b) is introduced into a plant having a sensitivity to high temperature stress:
(a) a protein comprising the amino acid sequence of SEQ ID NO: 2;
(b) a protein having an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 2, and having glutathione-S-transferase activity.

Description

Transgenic plants with high temperature stress resistance {High Temperature Resistant Transformed Plant}

The present invention relates to a method for imparting resistance to high temperature stress to a plant having a sensitivity to high temperature stress, and to a transgenic plant endowed with resistance to high temperature stress.

Compounds having an isoxazoline backbone (ie, isoxazoline derivatives) are known to have herbicidal activity. Also, "isoxazoline moiety" is also called "isooxazoline moiety". As isoxazoline derivatives, as described in Non-Patent Document 1, compounds such as pyroxasulfone have been developed that have excellent herbicidal effect and selectivity between crops and weeds. Pyroxasulfone acts on very-long-chain fatty acid elongase (VLCFAE), which catalyzes the biosynthesis of ultra-long-chain fatty acids, which are the main components of sphingolipids in the wax layer of plant epidermis and cell membranes. It is a herbicide with

Pyroxasulfone is known to be an upland crop herbicide that exhibits a high herbicidal effect against not only Gramineae weed and broadleaf weed, but also existing resistant weeds (Non-Patent Document 1). In particular, pyroxasulfone has safety in crops such as wheat. On the other hand, rapeseed, barley, rice, etc. are known to have high sensitivity to pyroxasulfone (Non-Patent Documents 1 and 2). Therefore, for rapeseed, barley, and rice, pyroxasulfone has not been registered as a herbicide and pesticide.

In addition, it is suggested that pyroxasulfone is related to drug metabolism by glutathione-S-transferase (GST) and resistance (wheat selectivity) in wheat (Non-Patent Document 3). In general, there have been many reports about the metabolism and detoxification by glutathione conjugation of pesticides (Non-Patent Documents 4 to 7). In particular, metabolism and detoxification by glutathione conjugation is known to be involved in the selectivity between crop weeds of chloroacetamide-based compounds, which are VLCFAE inhibitory herbicides, as well as pyroxasulfone (Non-Patent Documents 8 to 10).

GST is known to exist in several molecular species in plants. According to Non-Patent Document 11, corn-derived GST (GSTI, ZmM16901) exhibits high conjugation activity for chloroacetamide-based alachlor, whereas the structure is very similar to metolalachlor conjugation activity. It is disclosed that it does not represent In addition, Non-Patent Document 12 discloses that ZmGST8 (ZmQ9FQD1), which has the highest homology with ZmGSTI (the homology of amino acid sequence is about 56%), does not exhibit a conjugation activity to alachlor. As described above, since the substrate specificity of GST is high, it is very difficult to identify a molecular species of GST that metabolizes a specific drug, even if GST using a compound similar to the above-mentioned drug as a substrate is known.

In addition, plant GST directly reduces oxidative stress (Non-Patent Document 13) and is known to impart resistance to various stresses such as salt, drying temperature, and herbicides (Non-Patent Document 14), but herbicide tolerance and high temperature tolerance There are no reports of genes conferred at the same time. Moreover, although there is a report that high temperature tolerance was imparted to plants by introducing both GST and GPX (glutathione peroxidase) (Non-Patent Document 15), there is no report example that GST independently imparted high temperature tolerance.

Patent Document 1: US Patent US 6,730,828

Non-Patent Document 1: Y. Tanetani et al., Pestic. Biochem. Physiol. 95, 47-55 (2009) Non-Patent Document 2: Y. Tanetani et al., J. Pestic. Sci. 36, 221-228 (2011) Non-Patent Document 3: Y. Tanetani et al., J. Pestic. Sci. 38, 152-156 (2013) Non-Patent Document 4: E. Boyland et al., Adv. Enzymol. Relat. Areas. Mol. Biol. 32, 173-219 (1969) Non-Patent Document 5: B. Mannervik Adv. Enzymol. Relat. Areas. Mol. Biol. 57, 357-417 (1985) Non-Patent Document 6: PJ Hatton et al., Pestic. Sci. 46, 267-275 (1996). Non-Patent Document 7: DP Dixon et al., Genome Biol. 3, 1-10 (2002) Non-Patent Document 8: GL Lamoureux et al., J. Agric. Food Chem. 19, 346-350 (1971). Non-Patent Document 9: JRC Leavitt et al., J. Agric. Food. Chem. 27, 533-536 (1979). Non-Patent Document 10: T. Mozer et al., Biochemistry 22, 1068-1072 (1983) Non-Patent Document 11: M. Karavangeli et al., Biomolecular Engineering 22, 121 -128 (2005) Non-Patent Document 12: I. Cummins et al., Plant Mol. Biol. 52, 591-603 (2003) Non-Patent Document 13: I. Cummins et al., Plant J. 18, 285-292 (1999) Non-Patent Document 14: R Edwars & DP Dixson, Methods Enzymo l. 401, 169-186 (2005) Non-Patent Document 15: VP Roxas et al., Plant Cell Physiol. 41, 1229-1234 (2000)

Therefore, the present invention specifies glutathione-S-transferase that exhibits metabolic and detoxification activity against isoxazoline derivatives such as pyroxasulfone, and uses the glutathione-S-transferase to develop resistance to isoxazoline derivatives. An object of the present invention is to provide a method for using an isoxazoline derivative as a herbicide in a plant expressing the glutathione-S-transferase while providing a transgenic plant having

As a result of intensive studies conducted by the present inventors in order to achieve the above object, they succeeded in identifying glutathione-S-transferase present in wheat that exhibits metabolic and detoxifying activity against isoxazoline derivatives such as pyroxasulfone, and the glutathione The fact that the substrate specificity for the isoxazoline derivative of -S-transferase is very high, and surprisingly, it was discovered that the transgenic plant into which the glutathione-S-transferase (GST) was introduced acquires excellent stress tolerance, and the present invention came to complete.

The present invention includes the following.

(1) A method for cultivating a transgenic plant, characterized in that the transgenic plant into which the nucleic acid encoding the protein of (a) or (b) is introduced is grown in the presence of an isoxazoline derivative:

(a) a protein comprising the amino acid sequence of SEQ ID NO: 2;

(b) a protein having an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 2, and having glutathione-S-transferase activity.

(2) The cultivation method according to item (1), wherein the isoxazoline derivative is pyroxasulfone and/or phenoxasulfone.

(3) The cultivation method according to item (1), wherein the transgenic plant is derived from a plant having sensitivity to an isoxazoline derivative.

(4) The cultivation method according to the item (3), characterized in that the plant having a sensitivity to the isoxazoline derivative is a plant in the family Grapeaceae.

(5) The cultivation method according to the item (4), characterized in that the rice plant is rice.

(6) isoxazolyl sleepy plants with susceptible to derivatives cruciferous (Brassicaceae), leguminous (Leguminosae), Apiaceae (Umbelliferae), amaranth and (Amaranthaceae), Lamiaceae (Labiatae), chenopodiaceae (Chenopodiaceae), Rosaceae (Rosaceae ), Asteraceae ( Compositae ), Solanaceae ( Solanaceae ) or Malvaceae ( Malvaceae ) The cultivation method according to the item (3), characterized in that the plant.

(7) Cruciferous plants are rapeseed ( Brassica napus ) or Arabidopsis thaliana ( Arabidopsis thaliana ) The cultivation method according to item (6), characterized in that.

(8) A method for imparting resistance to an isoxazoline derivative, comprising introducing a nucleic acid encoding the protein of the following (a) or (b) into a plant having a sensitivity to the isoxazoline derivative:

(a) a protein comprising the amino acid sequence of SEQ ID NO: 2;

(b) a protein having an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 2, and having glutathione-S-transferase activity.

(9) The method for imparting resistance according to item (8), wherein the isoxazoline derivative is pyroxasulfone and/or phenoxasulfone.

(10) The method for imparting resistance according to the item (8), characterized in that the plant having a sensitivity to the isoxazoline derivative is a plant of the family Grapeaceae.

(11) The method for imparting tolerance according to the item (10), characterized in that the rice plant is rice.

12. isoxazolyl sleepy plants with susceptible to derivatives cruciferous (Brassicaceae), leguminous (Leguminosae), Apiaceae (Umbelliferae), amaranth and (Amaranthaceae), Lamiaceae (Labiatae), chenopodiaceae (Chenopodiaceae), Rosaceae (Rosaceae ), Asteraceae ( Compositae ), Solanaceae ( Solanaceae ) or Malvaceae ( Malvaceae ) The method for imparting resistance according to the item (8), characterized in that the plant.

(13) Cruciferous plants, rape ( Brassica napus ) or Arabidopsis thaliana ( Arabidopsis thaliana ) The method for imparting resistance according to the item (12), characterized in that.

(14) A method for imparting resistance to environmental stress, comprising introducing a nucleic acid encoding the protein of the following (a) or (b) to a plant:

(a) a protein comprising the amino acid sequence of SEQ ID NO: 2;

(b) a protein having an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 2, and having glutathione-S-transferase activity.

(15) The method for imparting resistance according to the item (14), wherein the environmental stress is high-temperature stress.

(16) The method for imparting tolerance according to item (14), wherein the plant is a plant having sensitivity to an isoxazoline derivative.

(17) The method for imparting resistance according to item (16), wherein the isoxazoline derivative is pyroxasulfone and/or phenoxasulfone.

(18) The method for imparting tolerance according to the item (14), characterized in that the plant is a plant in the family Grapeaceae.

(19) The method for imparting tolerance according to the item (18), characterized in that the rice plant is rice.

(20) Plant cruciferous (Brassicaceae), Mesquite (Leguminosae), Apiaceae (Umbelliferae), amaranthaceae (Amaranthaceae), Lamiaceae (Labiatae), chenopodiaceae (Chenopodiaceae), rose family (Rosaceae), Asteraceae (Compositae), Solanaceae ( Solanaceae ) or mallow family ( Malvaceae ) The method for imparting resistance according to the item (14), characterized in that the plant.

(21) Cruciferous plants are rapeseed ( Brassica napus ) or Arabidopsis thaliana ( Arabidopsis thaliana ) The method of imparting resistance according to the item (20), characterized in that.

(22) A transgenic plant into which a nucleic acid encoding the protein of (a) or (b) below has been introduced:

(a) a protein comprising the amino acid sequence of SEQ ID NO: 2;

(b) a protein having an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 2, and having glutathione-S-transferase activity.

(23) The transgenic plant according to item (22), characterized in that it has tolerance to isoxazoline derivatives and/or tolerance to environmental stress.

(24) The transgenic plant according to item (22), wherein the nucleic acid is introduced into a plant having a sensitivity to an isoxazoline derivative.

(25) The transgenic plant according to the item (23) or (24), wherein the isoxazoline derivative is pyroxasulfone and/or phenoxasulfone.

(26) The transgenic plant according to item (23), wherein the environmental stress is high temperature stress.

(27) The transgenic plant according to item (24), wherein the plant having a sensitivity to an isoxazoline derivative is a plant in the family Grapeaceae.

(28) The transgenic plant according to the item (28), characterized in that the plant of the family Grain is rice.

(29) isoxazolyl sleepy plants with susceptible to derivatives cruciferous (Brassicaceae), leguminous (Leguminosae), Apiaceae (Umbelliferae), amaranth and (Amaranthaceae), Lamiaceae (Labiatae), chenopodiaceae (Chenopodiaceae), Rosaceae (Rosaceae ), Asteraceae ( Compositae ), Solanaceae ( Solanaceae ) or Malvaceae ( Malvaceae ) The transgenic plant according to item (24), characterized in that the plant.

(30) The cruciferous plant is a transgenic plant, characterized in that rape ( Brassica napus ) or Arabidopsis thaliana ).

This specification includes the contents disclosed in the detailed description and/or drawings of Japanese Patent Application No. 2016-132689, which is the basis of the priority of the present application.

According to the present invention, there is provided a transgenic plant that has acquired resistance to isoxazoline derivatives such as pyroxasulfone and phenoxasulfone and/or resistance to environmental stress such as high temperature stress. That is, according to the present invention, to a plant having a sensitivity to the isoxazoline derivative, the isoxazoline derivative tolerance is imparted, and also environmental stress tolerance is imparted to the plant.

Therefore, since the transgenic plant according to the present invention can grow even in the presence of the isoxazoline derivative, stable cultivation and production is possible by using the isoxazoline derivative.

[FIG. 1] A schematic diagram showing the E. coli expression form of the TaQ8GTC0 protein.
[Figure 2] It is an electrophoresis photograph confirming the TaQ8GTC0 protein.
[Figure 3] Chemical formula of VLCFAE inhibitory herbicide tested for glutathione conjugation activity.
[Figure 4] Characteristic diagrams showing the conjugation reaction between pyroxasulfone and glutathione and HPLC charts when pyroxasulfone is used as a VLCFAE inhibitory herbicide.
[Fig. 5] A schematic diagram showing a vector for transformation of the TaQ8GTC0 gene into rice.
[Figure 6] It is a photograph showing the state in which the cultured cells of rice introduced with the TaQ8GTC0 gene were cultured in a selective medium.
[FIG. 7A] It is a characteristic diagram showing the state when line #3-13 and wild-type rice were grown in a medium containing a VLCFAE inhibitory herbicide.
[FIG. 7B] It is a characteristic diagram showing the state when line #3-13 and wild-type rice were grown in a medium containing a VLCFAE inhibitory herbicide.
[FIG. 7C] It is a characteristic diagram showing the state when line #3-13 and wild-type rice were grown in a medium containing a VLCFAE inhibitory herbicide.
[Fig. 7d] It is a characteristic diagram showing the state when line #3-13 and wild-type rice were grown in a medium containing a VLCFAE inhibitory herbicide.
[FIG. 7E] It is a characteristic diagram showing the state when line #3-13 and wild-type rice were grown in a medium containing a VLCFAE inhibitory herbicide.
[Fig. 7f] It is a characteristic diagram showing the state when line #3-13 and wild-type rice were grown in a medium containing a VLCFAE inhibitory herbicide.
[Figure 8] A schematic diagram showing a vector for transformation of Arabidopsis thaliana of the TaQ8GTC0 gene.
[FIG. 9] A photograph showing the state of culturing seeds collected from Arabidopsis plants into which the TaQ8GTC0 gene was introduced.
[Fig. 10] It is an electrophoresis photograph confirming the TaQ8GTC0 gene introduced into Arabidopsis.
[Figure 11] A characteristic diagram showing the test results of pyroxasulfone sensitivity to wild-type Arabidopsis thaliana (Columbia-0).
[Fig. 12] A characteristic diagram showing the results of the pyroxasulfone sensitivity test for Arabidopsis thaliana introduced with the TaQ8GTC0 gene.
[FIG. 13] A characteristic diagram showing the results of testing drug sensitivity to VLCFAE inhibitory herbicides for Arabidopsis thaliana introduced with TaQ8GTC0 gene.
[Fig. 14] A characteristic diagram showing the results of testing drug sensitivity to VLCFAE inhibitory herbicides for Arabidopsis thaliana introduced with TaQ8GTC0 gene.
[Fig. 15] A characteristic diagram showing the results of testing the high temperature stress tolerance for the TaQ8GTC0 transgenic rice.
[Fig. 16] A branching phylogenetic tree including GST with high homology to TaQ8GTC0 and TaQ8GTC0.
[Fig. 17] A schematic diagram showing a vector for rice transformation expressing various GSTs.
[FIG. 18] It is a characteristic diagram showing the results of measuring the fatty acid content in cultured cells of two types of plant GST gene-transduced rice cultured in a medium containing pyroxasulfonic acid, control rice culture cells, and control rice culture cells without drug treatment .
[FIG. 19] It is a characteristic diagram showing the results of measuring the fatty acid content in cultured cells of three types of plant GST gene-transduced rice cultured in a medium containing pyroxasulfonic acid, control rice culture cells, and control rice culture cells without drug treatment .
[Fig. 20] A characteristic diagram showing the results of measuring the fatty acid content in cultured cells of two types of plant GST transgenic rice cultured in a pyroxasulfonic acid-containing medium and cultured cells of control rice.
[Fig. 21] A schematic diagram showing a vector for transformation of rice expressing a maize GST gene.
[Fig. 22] It is a photograph showing the rice cultured cells into which the corn GST gene was introduced.
[Fig. 23] It is a photograph showing the result of testing the resistance to pyroxasulfone in the rice cultured cells introduced with the corn GST gene.
[Fig. 24] A schematic diagram showing the TaQ8GTC1-R gene replication.
[Fig. 25] It is a characteristic diagram showing the result of comparing the nucleotide sequence (upper row, SEQ ID NO: 31) of the TaQ8GTC1-R gene determined in this experiment with the nucleotide sequence (lower row, SEQ ID NO: 33) of the TaQ8GTC1 gene registered in the database .
[Figure 26] The result of comparing the amino acid sequence (upper row, SEQ ID NO: 32) encoded by the TaQ8GTC1-R gene determined in this experiment with the amino acid sequence (lower row, SEQ ID NO: 34) encoded by the TaQ8GTC1 gene registered in the database It is a characteristic diagram showing.
[ Fig. 27 ] A schematic diagram showing the E. coli expression form of the TaQ8GTC1-R protein.
[Fig. 28] An electrophoresis photograph confirming the TaQ8GTC1-R protein.
[Fig. 29] A characteristic diagram showing the HPLC chart when pyroxasulfone is used as a VLCFAE inhibitory herbicide and the conjugation reaction between pyroxasulfone and glutathione.
[FIG. 30] A characteristic diagram showing the result of comparing amino acid sequences between GSTs of various plants. First line: AtGSTF2 (SEQ ID NO: 37), second line: PttGSTF1 (SEQ ID NO: 38), third line: ZmGSTF1 (SEQ ID NO: 39), fourth line: TaGSTF1 (SEQ ID NO: 40), fifth line: TaGSTF2-R (SEQ ID NO: 32) ), line 6 : TaGSTF3 (SEQ ID NO: 2), line 7: TaGSTF4 (SEQ ID NO: 41), line 8: TaGSTF5 (SEQ ID NO: 42) and line 9: TaGSTF6 (SEQ ID NO: 43)
[Fig. 31] A schematic diagram showing a vector for transformation of the TaQ8GTC1-R gene into rice.
[FIG. 32] A photograph showing the state in which cultured cells of rice into which the TaQ8GTC1-R gene was introduced were selected and cultured in a selective medium.
[FIG. 33] A photograph showing the results of a tolerance test for pyroxasulfone of TaQ8GTC1-R transduced rice.
[FIG. 34] A schematic diagram showing a vector for transformation of the TaQ8GTC0 gene into rapeseed.
[FIG. 35] A photograph showing the state in which rapeseed cells into which the TaQ8GTC0 gene was introduced were selected and cultured.
[FIG. 36] It is a photograph of an electrophoresis picture showing the result of confirming by PCR whether the TaQ8GTC0 gene is introduced recombinant rapeseed.
[FIG. 37] A photograph showing the results of a soil treatment test before germination of pyroxasulfone for wild-type rapeseed (cultivar: Westar) and rapeseed with TaQ8GTC0 gene.
[Figure 38] A photograph showing the results of the pre-germination soil treatment test of pyroxasulfone for wild-type rape (cultivar: Westar) and rapeseed with TaQ8GTC0 gene.
[Figure 39] It is a photograph showing the results of the pre-germination soil treatment test of pyroxasulfone for wild-type rape (variety: Westar) and rapeseed with TaQ8GTC0 gene.
[FIG. 40] A photograph showing the results of a growth inhibition test in an agar medium for wild-type rapeseed (cultivar: Westar) and rapeseed with TaQ8GTC0 gene.
[FIG. 41] A photograph showing the results of a growth inhibition test on an agar medium for wild-type rape (variety: Westar) and rapeseed with TaQ8GTC0 gene.
[Fig. 42] A photograph showing the results of a growth inhibition test on an agar medium for wild-type rapeseed (cultivar: Westar) and rapeseed with TaQ8GTC0 gene.
[Fig. 43] A characteristic diagram showing the results of a growth inhibition test on an agar medium for wild-type rapeseed (cultivar: Westar) and rapeseed with TaQ8GTC0 gene.
[Figure 44] A characteristic diagram showing the results of a high temperature tolerance test in an agar medium for wild-type rape (variety: Westar) and rapeseed with TaQ8GTC0 gene.

Hereinafter, the present invention will be described in detail.

[Glutathione-S-transferase of the present invention]

The glutathione-S-transferase of the present invention (hereinafter, also abbreviated as "GST" for convenience) may be defined as the following (a) or (b) proteins:

(a) a protein comprising the amino acid sequence of SEQ ID NO: 2

(b) a protein having an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 2, and having glutathione-S-transferase activity

Here, the protein consisting of the amino acid sequence of SEQ ID NO: 2 is referred to as "GST F3" among GST derived from wheat (Triticum aestivum), and is specified by accession number: Q8GTC0. In addition, the amino acid sequence of SEQ ID NO: 2 is specified together with the CDS sequence (SEQ ID NO: 1) as accession number: AJ440792_1.

GST usable in the present invention is not limited to the protein consisting of the amino acid sequence of SEQ ID NO: 2, and as described in (b) above, 80% or more, preferably 85% or more, more preferably than the amino acid sequence of SEQ ID NO: 2 Preferably, it may be a protein having an amino acid sequence having an identity of 90% or more, more preferably 95% or more, and most preferably 97% or more, and having glutathione-S-transferase activity.

In addition, the value of amino acid sequence identity can be calculated by the BLASTN or BLASTX program which implemented the BLAST algorithm (default setting). In addition, the value of identity is calculated as a ratio among all amino acid residues compared by calculating exactly identical amino acid residues when a pair of amino acid sequences are analyzed for pairwise alignment.

In addition, GST usable in the present invention is not limited to the protein consisting of the amino acid sequence of SEQ ID NO: 2, has an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added to the amino acid sequence of SEQ ID NO: 2, glutathione A protein having -S-transferase activity may be used. Here, the number is, for example, 2 to 30, preferably 2 to 20, more preferably 2 to 15, still more preferably 2 to 10, and most preferably 2 to 5.

In addition, GST usable in the present invention is encoded by DNA that hybridizes under stringent conditions with respect to all or part of the complementary chain of DNA consisting of the nucleotide sequence of SEQ ID NO: 1, glutathione-S-transferase activity It may be any protein with "Stringent condition" means a condition under which a so-called specific hybrid is formed and a non-specific hybrid is not formed, for example, Molecular Cloning: A Laboratory Manual (Third Edition) ) and can be determined appropriately. Specifically, stringency may be set according to the temperature of Southern hybridization and the concentration of salt contained in the solution, and the temperature and concentration of salt contained in the solution during washing of Southern hybridization. More specifically, as stringent conditions, for example, a sodium concentration is 25-500 mM, Preferably it is 25-300 mM, and the temperature is 42-68 degreeC, Preferably it is 42-65 degreeC. More specifically, the salt concentration is 5xSSC (83 mM NaCl, 83 mM sodium citrate) and the temperature is 42°C.

In particular, GST usable in the present invention has glutathione-conjugation activity to isoxazoline derivatives, which will be described later. That is, GST usable in the present invention has an activity of binding an isoxazoline derivative and glutathione.

More specifically, when GST usable in the present invention is expressed in plants, it promotes glutathione conjugation to isoxazoline derivatives and reduces the activity inhibition of ultra-long-chain fatty acid elongation enzymes by isoxazoline derivatives. . Therefore, when the GST usable in the present invention is expressed in plants, it is possible to confer resistance to isoxazoline derivatives to the plants.

In other words, the above-described glutathione-S-transferase activity can be said to be glutathione-conjugating activity to isoxazoline derivatives or binding activity of glutathione to isoxazoline derivatives.

In addition, as described above, GST, which can also be used in the present invention for a protein containing an amino acid sequence different from the amino acid sequence of SEQ ID NO: 2, is an ultra-long-chain fatty acid elongation enzyme by an isoxazoline derivative in addition to the above-described glutathione-S-transferase activity. It may be GST having an action of reducing activity inhibition, and further, GST conferring resistance to isoxazoline derivatives to plants.

[transgenic plants]

The transgenic plant according to the present invention is introduced into a predetermined plant (plant, plant cell) so that the gene encoding the above-described GST can be expressed. The transgenic plant according to the present invention acquires resistance to an isoxazoline derivative and environmental stress to be described later by expressing the gene encoding the GST described above.

Here, the acquisition of resistance to the isoxazoline derivative means that the plant is subjected to the isoxazoline derivative after introduction of the above-described GST in comparison with the sensitivity of the plant to the isoxazoline derivative before the introduction of the above-described GST. A statistically significant decrease in susceptibility to That is, the acquisition of resistance to the isoxazoline derivative means that the inhibitory effect of the isoxazoline derivative on the plant after introducing the GST is statistically significant compared to the inhibitory effect on the plant before the GST is introduced. said to be lowered In addition, the inhibitory effect of the isoxazoline derivative can be evaluated using an index such as a 50% inhibitory concentration.

In addition, acquiring tolerance to environmental stress means that the susceptibility to the same environmental stress in the plant after introducing the above-described GST is statistically significantly reduced compared with the susceptibility of the plant to the environmental stress before introducing the above-mentioned GST. say to do That is, when culturing plants before introducing GST and plants after introducing the above-described GST under load conditions of a predetermined environmental stress to obtain environmental stress tolerance, the growth rate of plants after introducing GST is statistically Meaningly fast In addition, the growth rate of a plant may be evaluated using, for example, an indicator such as a length of a plant and a weight of a root.

Here, although environmental stress is not specifically limited, High temperature stress, high salt concentration stress, drying stress, and low temperature stress are mentioned. Among these various environmental stresses, the transgenic plant according to the present invention is particularly excellent in resistance to high temperature stress.

By the way, the target plant into which the above-mentioned GST is introduced is not particularly limited, and any plant may be used. Any plant can acquire environmental stress tolerance, such as high temperature stress tolerance, by introducing the above-mentioned GST. In particular, the target plant to introduce the above-described GST is preferably a plant having a sensitivity to an isoxazoline derivative to be described later. By introducing the above-described GST to a plant having a sensitivity to an isoxazoline derivative, the plant can acquire tolerance to the isoxazoline derivative.

Here, "plants having a sensitivity to an isoxazoline derivative" means a plant whose growth is inhibited at an optimal concentration when an isoxazoline derivative is used as a herbicide. For example, if the 50% inhibitory concentration (IC ₅₀ ) of the isoxazoline derivative for pyroxasulfone is 200 nM or less, preferably 100 nM or less, more preferably 50 nM or less, it is a plant having a sensitivity to pyroxasulfone. can do.

More specifically, plants with sensitivity to isoxazoline derivatives are not particularly limited, but include rice, barley, sorghum, oats, durum wheat, and Gramineae such as corn; Rape (Brassica rapa), rapeseed (Brassica napus), cabbage, canola, kale, white mustard (white mustard), turnip, such as Arabidopsis thaliana ( Brassicaceae ); leguminous plants such as alfalfa, bush bean, soybean, adzuki bean, mung bean, and shamrock ( Leguminosae ); Dill ( Anethum graveolens ), fennel ( fennel ), parsley and other buttercups ( Umbelliferae ) ; Spinach, sugar beet plants and the like amaranth (Amaranthaceae); Lamiaceae, such as basil ( Labiatae ) ; Cherries, etc. ( Chenopodiaceae ) ; Plums and other Rosaceae plants ( Rosaceae ), Asteraceae plants such as lettuce ( Compositae ); Solanaceae , such as tomatoes ( Solanaceae ); Malvaceae cotton, etc. can be enumerated plants (Malvaceae).

In this way, by introducing the above-described GST to the specifically exemplified plants, tolerance to the isoxazoline derivative can be imparted to the plant, and environmental stress tolerance can be improved.

In particular, the above-mentioned GST has high conjugation activity to isoxazoline derivatives among VLCFAE inhibitory herbicides, and is described in I. Cummins et al., Plant Mol. Biol. 52, 591-603 (2003), compared with the conjugation activity for metolachlor reported in statistically significantly higher. Therefore, the transgenic plants introduced with the above-described GST can be cultivated using isoxazoline derivatives among VLCFAE inhibitory herbicides as herbicides.

In addition, since the above-described transgenic plants introduced with GST are excellent in environmental stress tolerance, they can be cultivated even in an environment in which poor growth has occurred due to existing environmental stresses. In particular, the transgenic plants introduced with the above-described GST exhibit excellent resistance to high temperature stress among environmental stresses. Accordingly, the transgenic plants introduced with the above-described GST can be cultivated in a land and at a time unsuitable for growth due to a high existing temperature.

In addition, the method for constructing a transgenic plant according to the present invention is not particularly limited, and the above-described GST can be prepared by a method such as introducing an expression vector obtained by recombination of a nucleic acid encoding the GST described above into an expression vector into a plant. Transgenic plants that express can be constructed.

The expression vector is constructed to include a promoter that enables expression in plants and a nucleic acid encoding the above-mentioned GST. As a vector serving as a parent of the expression vector, various conventionally known vectors can be used. For example, a plasmid, phage, or cosmid may be used, and may be appropriately selected according to the introduced plant cell and the introduction method. Specifically, for example, pBR322, pBR325, pUC19, pUC119, pBluescript, pBluescriptSK, pBI-based vectors and the like can be used. In particular, when Agrobacterium is used as the method of introducing a vector into a plant, it is preferable to use a pBI-based binary vector. Specific examples of pBI-based binary vectors include pBIG, pBIN19, pBI101, pBI121, and the like.

The promoter is not particularly limited as long as it is a promoter capable of expressing the nucleic acid encoding the GST in a plant body, and a known promoter can be preferably used. In particular, it is preferable to use a constitutive expression promoter that constitutively expresses a downstream gene in a plant body as the promoter. Examples of the related promoter include cauliflower mosaic virus 35S promoter (CaMV35S), various actin gene promoters, various ubiquitin gene promoters, nopaline synthetase gene promoter, tobacco PR1a gene promoter, tomato ribulose 1 ,5-diphosphate carboxylase/oxidase small subunit gene promoter, napin gene promoter, and the like. Among these, the cauliflower mosaic virus 35S promoter, the actin gene promoter, or the ubiquitin gene promoter can be used more preferably. When each of the above promoters is used, any gene can be strongly expressed when introduced into plant cells.

In addition, a promoter having a function of site-specific expression of a plant may be used. As such a promoter, any conventionally known promoter may be used. Using such a promoter, the GST can be expressed site-specifically.

In addition, the expression vector may include another DNA fragment in addition to the promoter and the nucleic acid encoding the GST. The other DNA fragment is not particularly limited, but may include a terminator, a selectable marker, an enhancer, a nucleotide sequence for increasing translation efficiency, and the like. In addition, the expression vector may have a T-DNA region. In particular, the T-DNA region can increase gene introduction efficiency when the recombinant expression vector is introduced into a plant using Agrobacterium.

A transcription terminator is not particularly limited as long as it has a function as a transcription termination site, and a known one may be used. For example, specifically, the transcription termination region (Nos terminator) of the nopaline synthetase gene, the cauliflower mosaic virus 35S transcription termination region (CaMV35S terminator), etc. can be preferably used. Among these, the Nos terminator can be used more preferably. In the expression vector, it is possible to prevent the synthesis of unnecessarily long transcripts after introduction into plant cells by arranging the transcription terminator at an appropriate position.

As a transformant selection marker, for example, a drug resistance gene can be used. Specific examples of the related drug resistance gene include, for example, drug resistance genes to hygromycin, bleomycin, kanamycin, gentamicin, chloramphenicol, and the like. Therefore, the transformed plant can be easily selected by selecting the plant growing in the medium containing the antibiotic. In addition, as the transformant selection marker, a mutant acetolactate synthase gene that confer resistance to a predetermined drug may be used.

The method for constructing the expression vector is not particularly limited, and the promoter and the nucleic acid encoding the GST and, if necessary, the other DNA fragments may be introduced in a predetermined order into an appropriately selected parent vector. For example, an expression cassette is constructed by linking the GST-encoding nucleic acid with a promoter (eg, a transcription terminator if necessary), and this may be introduced into a vector. Construction of the expression cassette makes it possible to define the order of the DNA fragments, for example, by placing the cleavage sites of each DNA fragment at complementary protruding ends and reacting them with a ligase. In the case where the terminator is included in the expression cassette, the order of the promoter, the GST-encoding nucleic acid, and the terminator from the upstream is sufficient. In addition, the type of reagent for constructing the expression vector, ie, restriction enzyme and binding enzyme, is not particularly limited, and commercially available ones may be appropriately selected and used.

In addition, the propagation method (production method) of the expression vector is not particularly limited, and a conventionally known method can be used. In general, E. coli as a host may be propagated in the E. coli. At this time, a preferred type of E. coli is selected according to the type of vector.

The above-described expression vector is introduced into the target plant by a general transformation method. A method (transformation method) for introducing an expression vector into a plant cell is not particularly limited, and a conventionally known method suitable for the plant cell may be used. Specifically, for example, a method using Agrobacterium and a method of direct introduction into plant cells can be used. Methods for introducing the expression vector directly into plant cells include, for example, microinjection, electroporation, polyethylene glycol method, particle gun method, and protoplast fusion method. (protoplast fusion), calcium phosphate method (calcium phosphate method), etc. can be used.

In addition, if the method of introducing DNA directly into plant cells is adopted, it is sufficient if the DNA containing the transcriptional units necessary for expression of the target gene, for example, a promoter and a transcriptional terminator, and a nucleic acid encoding the GST is sufficient. and the vector function is not required. In addition, even a DNA including only the coding region of the GST without a transcription unit can be used as long as it can be integrated into the transcription unit of the host to express the target GST.

Plant cells into which the expression vector and the expression cassette containing the nucleic acid encoding the GST to be excluded are introduced include, for example, cells of each tissue in plant organs such as flowers, leaves, and roots, callus, Suspension cultured cells etc. are mentioned. Here, the expression vector may be appropriately constructed according to the type of plant to be produced, but a general expression vector may be established in advance and introduced into plant cells.

The tumor tissue, shoot, hairy root, etc. obtained as a result of transformation can be used for cell culture, tissue culture or organ culture by itself, and also known plants Plant hormones (auxin, cytokine, gibberellin, abscisic acid, ethylene, brassinoride, etc.) of appropriate concentrations using tissue culture method The plant can be regenerated by administration or the like.

The regeneration method adopts a method of obtaining a complete plant by transferring the callus-shaped transformed cells to a medium with different hormone types and concentrations, culturing, and forming adventitious embryos. As a medium to be used, LS medium, MS medium, etc. are illustrated.

In addition, in the transgenic plant according to the present invention, a transformed plant cell is obtained by introducing an expression vector containing the nucleic acid encoding the GST into a host cell, and the transformed plant cell is regenerated from the transformed plant cell. A plant seed obtained from a plant body and a later plant obtained from the plant seed are also included. To obtain plant seeds from a transgenic plant, for example, a transgenic plant is transplanted into a pot containing soil containing water collected from a rooting medium and grown at a constant temperature to form flowers to form seeds. make it In addition, in order to produce a plant from a seed, for example, a seed formed from a transgenic plant is isolated from a mature place, sown in soil containing water, and grown under a constant temperature and illumination to produce a plant. The plant thus constructed exhibits tolerance to isoxazoline derivatives and tolerance to environmental stress in order to express the above-described GST.

[Isoxazoline derivatives]

In the present invention, the isoxazoline derivative includes a compound having an isoxazoline skeleton and a salt thereof. The herbicidal activity of isoxazoline derivatives has been reported, for example, in Japanese Patent Application Laid-Open No. 8-225548, Japanese Patent Application Laid-Open No. 9-328477, and Japanese Patent Application Laid-Open No. 9-328483, and these previously reported isoxazoline derivatives. Zoline derivatives can be used as herbicides.

In addition, Patent No. 4465133 discloses a group of compounds having a herbicidal effect and selectivity between crops and weeds among isoxazoline derivatives. The isoxazoline derivative disclosed in Patent No. 4465133 is pyroxasulfone (compound name: 3-[5-(difluoromethoxy)-1-methyl-3-(trifluoromethyl)pyrazol-4-ylmethyl) sulfonyl-4,5-dihydro-5,5-dimethyl-1, 2-oxazole).

Specifically, Patent No. 4465133 discloses isoxazoline derivatives described in the following items (1) to (17).

(1) an isoxazoline derivative represented by the general formula [I] or a pharmacologically acceptable salt thereof:

In the above formula,

R ¹ and R ² are the same or different, and each represents a hydrogen atom, a C1-C10 alkyl group, a C3-C8 cycloalkyl group, or a C3-C8 cycloalkyl C1-C3 alkyl group, or R ¹ and R ² are bonded to each other to bond them; together with a C3-C7 spiro ring;

R ³ and R ⁴ are the same or different, and each represents a hydrogen atom, a C1-C10 alkyl group or a C3-C8 cycloalkyl group, or R ³ and R ⁴ are bonded to each other and together with the carbon atom to which they are bonded, a C3-C7 spiro ring or R ¹ , R ² , R ³ and R ⁴ may form a 5-8 membered ring together with the carbon atom to which they are attached;

R ⁵ and R ⁶ are the same or different and each represents a hydrogen atom or a C1-C10 alkyl group;

Y represents a 5- to 6-membered aromatic heterocyclic group or a condensed aromatic heterocyclic group having an arbitrary hetero atom selected from a nitrogen atom, an oxygen atom and a sulfur atom, and these heterocyclic groups are described later may be substituted with 0 to 6 identical or different groups selected from the substituent group α, and adjacent alkyl groups, alkoxy groups, alkyl groups and alkoxy groups, alkyl groups and alkylthio groups, alkyl groups and alkylsulfonyl groups, alkyl groups and monoalkylamino groups , or an alkyl group and two dialkylamino groups may be combined to form a 5 to 8 membered ring substitutable with 1 to 4 halogen atoms, and, in the case where the heteroatom of these heterocyclic groups is a nitrogen atom, it is oxidized to form an N-oxide It is good to do; and,

n represents the integer of 0-2.

Of the following substituent groups, "a substitutable phenyl group, a substitutable phenoxy group, a substitutable phenylthio group, a substitutable aromatic heterocyclic group, a substitutable aromatic heterocyclic oxy group, a substitutable aromatic heterocyclic thio group, a substitutable phenylsulfinyl group, a substitutable Phenylsulfonyl group, substitutable aromatic heterocyclic sulfonyl group, substitutable phenylsulfonyloxy group, substitutable benzylcarbonyl group, substitutable benzoyl group, substitutable benzyloxycarbonyl group, substitutable phenoxycarbonyl group, substitutable benzylcarbonyloxy group , or a substitutable benzoyloxy group” means that the substituent is a halogen atom, a C1-C10 alkyl group, a C1-C4 haloalkyl group, a C1-C10 alkoxyalkyl group, a C1-C10 alkoxy group, a C1-C10 alkyl group a group, a C1-C10 alkylsulfonyl group, an acyl group, a C1-C10 alkoxycarbonyl group, a cyano group, a carbamoyl group (the nitrogen atom of the group may be substituted with the same or different C1-C10 alkyl groups), a nitro group or an amino group ( The nitrogen atom of the group may be substituted with the same or different C1-C10 alkyl group, C1-C6 acyl group, C1-C4 haloalkylcarbonyl group, C1-C10 alkylsulfonyl group, or C1-C4 haloalkylsulfonyl group) It indicates that it may be substituted.

"Substituent group α"

A hydroxyl group, a thiol group, a halogen atom, a C1-C10 alkyl group, a C1-C10 alkyl group monosubstituted with an arbitrary group selected from the substituent group β, a C1-C4 haloalkyl group, a C3-C8 cycloalkyl group, a C1-C10 alkoxy group, a substituent group C1-C10 alkoxy group, C1-C4 haloalkoxy group, C3-C8 cycloalkyloxy group, C3-C8 cycloalkyl C1-C3 alkyloxy group, C1-C10 alkylthio group monosubstituted with any group selected from γ; C1-C10 alkylthio group, C1-C4 haloalkylthio group, C2-C6 alkenyl group, C2-C6 alkenyloxy group, C2-C6 alkynyl group, C2-C6 alkyl group monosubstituted with any group selected from the substituent group γ C1-C10 alkylsulfinyl group monosubstituted with an optional group selected from nyloxy group, C1-C10 alkylsulfinyl group, substituent group γ, C1-C10 alkylsulfonyl group, C1-C10 monosubstituted with an optional group selected from the substituent group γ C10 alkylsulfonyl group, C1-C4 haloalkylsulfinyl group, C1-C10 alkylsulfonyloxy group monosubstituted with an arbitrary group selected from the substituent group γ, C1-C4 haloalkylsulfonyl group, C1-C10 alkylsulfonyloxy group, C1 ~C4 Haloalkylsulfonyloxy group, substitutable phenyl group, substitutable phenoxy group, substitutable phenylthio group, substitutable aromatic heterocyclic group, substitutable aromatic heterocyclic oxy group, substitutable aromatic heterocyclic thio group, substitutable phenylsulfinyl group , Substitutable phenylsulfonyl group, substitutable aromatic heterocyclic sulfonyl group, substitutable phenylsulfonyloxy group, acyl group, C1-C4 haloalkylcarbonyl group, substitutable benzylcarbonyl group, substitutable benzoyl, carboxyl group, C1-C10 alkoxycarbo A nyl group, a substitutable benzyloxycarbonyl group, a substitutable phenoxycarbonyl group, a cyano group, a carbamoyl group (the nitrogen atoms of the groups may be the same or different, and may be substituted with a C1~C10 alkyl group or a substitutable phenyl group), C1~C6 Acyloxy group, C1-C4 haloalkylcarbonyloxy group, substitutable benzylcarbonyloxy group, substitutable benzoyloxy group nitro group, amino group (the nitrogen atoms of the groups are the same or different, C1-C10 alkyl group, substitutable phenoxy group Nyl group, C1~C6 acyl group, C1~C4 haloalkylcarbonyl group, substitutable benzylcarbonyl group, substitutable benzoyl group, C1~C10 alkylsulfonyl group, C1~C4 haloalkylsulfonyl group, substitutable benzylsulfonyl group or substitutable It may be substituted with a phenylsulfonyl group).

"Substituent group β"

hydroxyl group, C3-C8 cycloalkyl group (the group may be substituted with a halogen atom or an alkyl group), C1-C10 alkoxy group, C1-C10 alkylthio group, C1-C10 alkylsulfonyl group, C1-C10 alkoxycarbonyl group, C2 -C6 haloalkenyl, amino group (the nitrogen atom of the group is the same or different, C1~C10 alkyl group, C1~C6 acyl group, C1~C4 haloalkylcarbonyl group, C1~C10 alkylsulfonyl group, C1~C4 haloalkylsulfonyl may be substituted), a carbamoyl group (the nitrogen atom of the group may be substituted with the same or different C1-C10 alkyl groups), C1-C6 acyl group, C1-C4 haloalkylcarbonyl group, C1-C10 alkoxyimino group , a cyano group, a substituted phenyl group, a substituted phenoxy group.

"Substituent group γ"

A C1~C10 alkoxycarbonyl group, a substitutable phenyl group, a substitutable aromatic heterocyclic group, a cyano group, and a carbamoyl group (the nitrogen atoms of the groups may be substituted with the same or different C1~C10 alkyl groups).

(2) C1~C10 alkyl group, C1~C4 halo monosubstituted with any group selected from hydroxyl group, halogen atom, C1~C10 alkyl group, substituent group β Alkyl group, C3-C8 cycloalkyl group, C1-C10 alkoxy group, C1-C10 alkoxy group monosubstituted with an arbitrary group selected from the substituent group γ, C1-C4 haloalkoxy group, C3-C8 cycloalkyloxy group, C3-C8 Cycloalkyl C1~C3 alkyloxy group, C1~C10 alkylthio group, C1~C10 alkylthio group substituted with any group selected from the substituent group γ, C1~C4 haloalkylthio group, C2-C6 alkenyl group, C2- C6 alkenyloxy group, C2-C6 alkynyl group, C2-C6 alkynyloxy group, C1-C10 alkylsulfonyl group, C1-C4 haloalkylsulfonyl group, substitutable phenyl group, substitutable phenoxy group, substitutable phenylthio group, substitutable Aromatic heterocyclic group, substitutable aromatic heterocyclic oxy group, substitutable aromatic heterocyclic thio group, substitutable phenylsulfonyl group, substitutable aromatic heterocyclic sulfonyl group, C1-C6 acyl group, C1-C4 haloalkylcarbonyl group, substitutable Benzylcarbonyl group, substitutable benzoyl, carboxyl group, C1~ C10 alkoxycarbonyl group, cyano group, carbamoyl group (the nitrogen atom of the group may be the same or different, and may be substituted with a C1~ C10 alkyl group or a substitutable phenyl group), nitro group , amino group (the nitrogen atom of the group is the same or different, C1~C10 alkyl group, substitutable phenyl group, C1~C6 acyl group, C1~C4 haloalkylcarbonyl group, substitutable benzylcarbonyl group, substitutable benzoyl group, C1~C10 alkyl may be substituted with a sulfonyl group, a C1-C4 haloalkylsulfonyl group, a substitutable benzylsulfonyl group or a substitutable phenylsulfonyl group), or adjacent alkyl groups, alkoxy groups, an alkyl group and an alkoxy group, an alkyl group and an alkylthio group; The group according to the above (1), wherein an alkyl group and an alkylsulfonyl group, an alkyl group and a monoalkylamino group or two alkyl groups and a dialkylamino group may be combined to form a 5-8 membered ring substituted with 1 to 4 halogen atoms. Reconstituted isoxazoline derivatives.

(3) Substituent group α of a heterocyclic ring that can be substituted with 0-6 identical or different groups is a halogen atom, a C1-C10 alkyl group, a C1-C4 haloalkyl group, a C1-C10 alkoxy C1-C3 alkyl group, a C3-C8 cycloalkyl group (these groups are halogen atom or alkyl group), C1~C10 alkoxy group, C1~C4 haloalkoxy group, C3-C8 cycloalkyl C1~C3 alkyloxy group, substitutable phenoxy group, C1~C10 alkylthio group, C1~ C10 alkylsulfonyl group, acyl group, C1-C4 haloalkylcarbonyl group, C1-C10 alkoxycarbonyl group, cyano group or carbamoyl group (the nitrogen atom of the group may be substituted with the same or different C1-C10 alkyl groups) (2) The isoxazoline derivative according to the item.

(4) R ¹ and R ² are the same or different, a methyl group or an ethyl group, and R ³ , R ⁴ , R ⁵ and R ⁶ are isoxanes according to the item (1), (2) or (3), wherein R 3 , R 4 , R 5 and R 6 are hydrogen atoms. sleepy derivatives.

(5) The above-mentioned (1), (2), (3) or (4), wherein Y is a 5-membered or 6-membered aromatic heterocyclic group having an arbitrary hetero atom selected from a nitrogen atom, an oxygen atom and a sulfur atom. Isoxazoline derivatives.

(6) The isoxazoline derivative according to the above (5), wherein Y is a thienyl group, a pyrazolyl group, an isoxazolyl group, an isothiazolyl group, a pyridyl group or a pyrimidinyl group.

(7) Y is a thiophen-3-yl group, a pyrazol-4-yl group, a pyrazol-5-yl group, an isoxazol-4-yl group, an isothiazol-4-yl group, a pyridin-3-yl group or a pyrimidine- The isoxazoline derivative according to the item (6), which is a 5-yl group.

(8) The isoxazoline derivative according to the above (7), wherein Y is a thiophen-3-yl group, and positions 2 and 4 of the thiophene ring are necessarily substituted with a substituent group α.

(9) Y is a pyrazol-4-yl group, positions 3 and 5 of the pyrazole ring are necessarily substituted with a substituent group α, and position 1 is necessarily selected from a hydrogen atom, a C1-C10 alkyl group, and a substituent group β C1~C10 alkyl group monosubstituted with any group, C1~C4 haloalkyl group, C3-C8 cycloalkyl group, C2-C6 alkenyl group, C2-C6 alkynyl group, C1~C10 alkylsulfinyl group, C1~C10 alkylsulfonyl group, substituent A C1-C10 alkylsulfonyl group monosubstituted with any group selected from the group γ, a C1-C4 haloalkylsulfonyl group, a substitutable phenyl group, a substitutable aromatic heterocyclic group, a substitutable phenylsulfonyl group, a substitutable aromatic heterocyclic sulfonyl group, Acyl group, C1~C4 haloalkylcarbonyl group, substitutable benzylcarbonyl group, substitutable benzoyl group, C1~C10 alkoxycarbonyl group, substitutable benzyloxycarbonyl group, substitutable phenoxycarbonyl group, carbamoyl group (of The nitrogen atom may be substituted with the same or different, C1-C10 alkyl group or a substitutable phenyl group), an amino group (the nitrogen atom of the group is the same or different, a C1-C10 alkyl group, a substituted phenyl group, an acyl group, a C1-C4 haloalkyl carbo The above (which may be substituted with a substituted benzylsulfonyl group or a substitutable phenylsulfonyl group) The isoxazoline derivative according to item 7).

(10) Y is a pyrazol-5-yl group, the 4th position of the pyrazole ring is necessarily substituted with a substituent group α, and the 1st position is necessarily selected from a hydrogen atom, a C1-C10 alkyl group, and a substituent group β. C1-C10 alkyl group monosubstituted with a group, C1-C4 haloalkyl group, C3-C8 cycloalkyl group, C2-C6 alkenyl group, C2-C6 alkynyl group, C1-C10 alkylsulfinyl group, C1-C10 alkylsulfonyl group, substituent group γ C1-C10 alkylsulfonyl group monosubstituted with any group selected from, C1-C4 haloalkylsulfonyl group, substitutable phenyl group, substitutable aromatic heterocyclic group, substitutable phenylsulfonyl group, substitutable aromatic heterocyclic sulfonyl group, acyl group , C1~ C4 haloalkylcarbonyl group, substitutable benzylcarbonyl group, substitutable benzoyl group, C1~C10 alkoxycarbonyl group, substitutable benzyloxycarbonyl group, substitutable phenoxycarbonyl group, carbamoyl group (the nitrogen atom of the group is The same or different C1~C10 alkyl groups or substitutable phenyl groups may be substituted), amino groups (the nitrogen atoms of the groups are the same or different, C1~C10 alkyl groups, substitutable phenyl groups, acyl groups, C1~C4 haloalkylcarbonyl groups, substituted Item (7) above which may be substituted with a possible benzylcarbonyl group, a substitutable benzoyl group, a C1-C10 alkylsulfonyl group, a C1-C4 haloalkylsulfonyl group, a substitutable benzylsulfonyl group, or a substitutable phenylsulfonyl group) isoxazoline derivatives described in

(11) The isoxazoline derivative according to the above (7), wherein Y is an isoxazol-4-yl group, and the 3rd and 5th positions of the isoxazole ring are necessarily substituted with a substituent group α.

(12) The isoxazoline derivative according to the above (7), wherein Y is an isothiazol-4-yl group, and the 3rd and 5th positions of the isothiazole ring are necessarily substituted with a substituent group α.

(13) The isoxazoline derivative according to the above (7), wherein Y is a pyridin-3-yl group, and the 2-position and 4-position of the pyridine ring are necessarily substituted with a substituent group α.

(14) The isoxazoline derivative according to the above (7), wherein Y is a pyrimidin-5-yl group, and the 4th and 6th positions of the pyrimidine ring are necessarily substituted with a substituent group α.

(15) The isoxazoline derivative according to any one of (1) to (14), wherein n is an integer of 2.

(16) The isoxazoline derivative according to any one of (1) to (14), wherein n is the integer 1.

(17) The isoxazoline derivative according to any one of (1) to (14), wherein n is an integer 0.

In addition, Patent No. 4299483 also discloses a group of compounds having a herbicidal effect and selectivity between crops and weeds among isoxazoline derivatives. The isoxazoline derivative disclosed in Patent No. 4299483 is phenoxasulfone (compound name: 3-[(2,5-dichloro-4-ethoxybenzyl)sulfonyl]-4,5-dihydro-5,5- dimethyl-1,2-oxazole).

Specifically, Patent No. 4299483 discloses the isoxazoline derivatives described in the following items (18) to (20) and salts thereof.

(18) Isoxazoline derivatives represented by the general formula [I] and salts thereof.

In the above formula,

Q represents the group -S(O)n-(CR ₅ R ₆ )m-, n represents an integer of 0 to 2, m represents an integer of 1 to 3, R ₅ and R ₆ each independently represent a hydrogen atom, a cyano group, an alkoxycarbonyl group, or a C1-C6 alkyl group;

R ₁ and R ₂ are hydrogen atoms (provided that R ₁ and R ₂ cannot be hydrogen atoms at the same time), [C3-C8 cycloalkyl group, C1-C6 alkoxy group, C1-C6 alkylcarbonyl group, C1-C6 alkylthi Group, C1~C6 alkylsulfinyl group, C1-6 alkylsulfonyl group, C1~C6 alkylamino group, di(C1~C6 alkyl)amino group, cyano group, C1~C6 alkoxycarbonyl group, C1~C6 alkylaminocarbonyl group, di (C1~C6 alkyl)aminocarbonyl group, (C1~C6 alkylthio)carbonyl group, carboxyl group, benzyloxy group (which may be substituted by 1 to 5 halogen atoms, C1~C6 alkyl groups or C1~C6 alkoxy groups), ( A phenoxy group (which may be optionally substituted with 1 to 5 halogen atoms, C1 to C6 alkyl groups or C1 to C6 alkoxy groups) or a phenyl group (which may be substituted with 1 to 5 halogen atoms, C1 to C6 alkyl or C1 to C6 alkoxy groups) which may be substituted] C1~C8 alkyl group, C3-C8 cycloalkyl group, C1~C6 alkoxycarbonyl group, C1~C6 alkylaminocarbonyl group, di(C1~C6 alkyl)aminocarbonyl group, (C1~C6 alkylthio)carbo represents a nyl group, a carboxyl group, or a phenyl group (which may be substituted with 1 to 5 halogen atoms, C1 to C6 alkyl or C1 to C6 alkoxy groups), or R ₁ and R ₂ together with their bonding carbon atoms are C3-C7 spiro can form a ring;

R ₃ and R ₄ represent a hydrogen atom, a C1-C8 alkyl group or a C3-C8 cycloalkyl group (which may be substituted with 1 to 3 halogen atoms, a C3-C8 cycloalkyl group or a C1-C6 alkoxy group, identical or different), R ₃ and R ₄ together with their bonding carbon atoms may form a C3-C7 spiro ring, or R ₁ , R ₂ , R ₃ and R ₄ together with their bonding carbon atoms form a 5-8 membered ring. may be formed;

Y is a hydrogen atom, C1-C6 alkoxycarbonyl group, C2-C6 alkenyl group, [same or different, 1 to 3 halogen atoms, C1-C6 alkoxy group, C2-C6 alkenyloxy group, C2-C6 alkynyloxy group ( Halogen atom, C1~C6 alkyl or C1~C6 alkoxy group which may be substituted by 1 to 5) benzyloxy group, C1~C6 alkoxycarbonyl group, carboxyl group, hydroxyl group or methionine group] C1~C10 alkyl group or ( 1-5 identical or different R ₇ ) substituted) phenyl group; and,

R ⁷ is a hydrogen atom, [same or different, 1 to 3 halogen atoms, C1 to C6 alkoxy group, hydroxyl group, C1 to C6 alkylthio group, C1 to C6 alkylsulfinyl group, C1 to C6 alkylsulfonyl group, C1 to C6 Alkylamino group, di(C1~C6)alkylamino group, cyano group, or a C1~C6 alkyl group (which may be substituted with 1 to 5 halogen atoms, C1~C6 alkyl or C1~C6 alkoxy groups may be substituted with phenoxy)] , (same or different, 1 to 3 halogen atoms, C1~C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group, C1~C6 alkoxycarbonyl group, C1~C6 alkylcarbonyl group or C3-C8 cycloalkyl group C1~C6 alkoxy group, C2-C6 alkenyl group, C3-C8 cycloalkyloxy group (which may be substituted with 1 to 3 halogen atoms or C1~C6 alkoxy group, which may be the same or different) C1~C6 Alkylthio group, (same or different, which may be substituted with 1 to 3 halogen atoms or C1 to C6 alkoxy groups) C1 to C6 alkylsulfinyl group, (same or different, 1 to 3 halogen atoms or C1 to C6 alkoxy groups) optionally substituted C1~C6 alkylsulfonyl group, (halogen atom, 1~5 C1~C6 alkyl or C1~C6 alkoxy groups which may be substituted) benzyloxy group, (C1~C6 alkyl group, C1~C6 alkylsulfo) nyl group, C1-C6 alkylcarbonyl (C1-C6 alkyl) group, or C1-C6 alkylsulfonyl (C1-C6 alkyl) group) amino group, di(C1-C6 alkyl) amino group, halogen atom, cyano group , nitro group, C 1 to C6 alkoxycarbonyl group, C3-C8 cycloalkyloxycarbonyl group, carboxyl group, C2-C6 alkenyloxycarbonyl group, C2-C6 alkynyloxycarbonyl group (halogen atom, C1-C6 alkyl or C1 ~ C6 alkoxy group may be substituted by 1 to 5) benzyloxycarbonyl group (halogen atom, C1 to C6 alkyl, or 1 to 5 C1 to C6 alkoxy groups may be substituted) phenoxycarbonyl group or C1 to C6 alkylcarbo represents a nyloxy group.

(19) Isoxazoline derivatives represented by the general formula [I] in the above (18) and salts thereof:

In the above formula,

Q represents -S(O)n-(CR ₅ R ₆ )m- (wherein n is an integer of 0 to 2, m is 1, and R ₅ and R ₆ are hydrogen atoms);

R ₁ and R ₂ are hydrogen atoms (provided that R ₁ and R ₂ cannot be hydrogen atoms at the same time), (which may be substituted with a C3-C8 cycloalkyl group or a C1-C6 alkoxy group) C1-C8 alkyl group or C3-C8 represents a cycloalkyl group, or R ₁ and R ₂ together with their bonding carbon atoms may form a C3-C7 spiro ring;

R ₃ and R ₄ represent a hydrogen atom or a C1-C8 alkyl group (which may be substituted with 1 to 3 halogen atoms, a C3-C8 cycloalkyl group or a C1-C6 alkoxy group, identical or different, and R ₃ and R ₄ are may form a C3-C7 spiro ring together with these bonding carbon atoms, or R ₁ , R ₂ , R ₃ and R ₄ may form a 5-8 membered ring together with their bonding carbon atoms;

Y represents a phenyl group (substituted with 1-5 identical or different R _{7 );}

R ₇ is a hydrogen atom, [same or different, 1 to 3 halogen atoms, C1 to C6 alkoxy group, hydroxyl group, C1 to C6 alkylthio group, C1 to C6 alkylsulfinyl group, C1 to C6 alkylsulfonyl group, C1 to C6 An alkylamino group, a di(C1~C6)alkylamino group, a cyano group, or a phenoxy group (which may be substituted with 1 to 5 halogen atoms, C1~C6 alkyl or C1~C6 alkoxy groups) C1~C6 alkyl group , (same or different, 1 to 3 halogen atoms, C1~C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group, C1~C6 alkoxycarbonyl group, C1~C6 alkyl carbonyl group or C3-C8 cycloalkyl group may be substituted) C1~C6 alkoxy group, C3-C8 cycloalkyloxy group or halogen atom.

(20) Isoxazoline derivatives represented by the general formula [I] in the above (18) and salts thereof:

In the above formula,

Q represents -S(O)n-(CR ₅ R ₆ )m- (wherein n is an integer of 0 to 2, m is 1, and R ₅ and R ₆ are hydrogen atoms);

R ₁ and R ₂ represent a C1-C8 alkyl group;

R ₃ and R ₄ represent a hydrogen atom;

Y represents a phenyl group (substituted with 1-5 identical or different R _{7 );} and,

R ₇ is a hydrogen atom, a C1~C6 alkyl group (same or different, which may be substituted with 1 to 3 halogen atoms or C1~C6 alkoxy groups), (same or different, 1 to 3 halogen atoms or C1~C6 alkoxy groups) optionally substituted) C1-C6 alkoxy group, or a halogen atom.

The above-mentioned GST has a conjugation activity to the previously reported isoxazoline derivatives. Accordingly, the transgenic plants introduced with GST have resistance to these previously reported isoxazoline derivatives.

In particular, the above-mentioned GST is an isoxazoline derivative disclosed in Patent No. 4465133 (that is, the isoxazoline derivative described in items (1) to (17) above) and an isoxazoline derivative disclosed in Patent No. 4299483 (that is, , has a very good conjugation activity against the isoxazoline derivatives described in the above (18) to (20)). Accordingly, the transgenic plants introduced with the GST described above have further increased resistance to the isoxazoline derivative disclosed in Patent No. 4465133 and the isoxazoline derivative disclosed in Patent No. 4299483.

Moreover, the above-mentioned GST has very good conjugation activity with respect to pyroxasulfone and phenoxasulfone. Accordingly, the transgenic plants introduced with the above-described GST have further increased resistance to pyroxasulfone and phenoxasulfone.

When the above-described isoxazoline derivative is used as a herbicide, it is possible to control the growth of various weeds except for the transgenic plants introduced with the above-described GST. These weeds are not particularly limited, but for example, Echinochloa crus-galli ( L. ) Beauv. var. crus-galli ), Digitaria ciliaris ( Retz. ) Koeler ), Foxtail ( Setaria viridis ( L. ) . ) Beauv. ), Poa annua L. , Sorghum halepense ( L. ) Pers. ), Alopecurus aequalis Sobol. var. amurensis ( Komar. ) Ohwi ), wild oats (wild) oats), such as Gramineae weeds (Gramineae weed), as well as the, huinyeokkwi (Polygonum lapathifolium L. nodosum (Pers. ) Kitam.), Zheng amaranth (Amaranthus viridis L.), goosefoot (Chenopodium album L.), chickweed (Stellaria media ( L. ) Villars ), Swallowtail ( Abutilon avicennae ), satin grass ( Sida spinosa ), C assia obtusifolia , Lagium ( Ambrosia artemisiifolia L. var. elatior ( L. ) Desc. ), morning glory broadleaf weed, Cyperus rotundus L. , cyperus esculentus , Kyllinga brevifolia Rottb. subsp. leiolepis ( Fraxch. et Savat. ) T. Koyama ), Cyperus microiria Steud. ), and Cyperus iria L. ) and other perennial or annual cyperaceous weeds. In addition, when isoxazoline derivatives are used in paddy fields as herbicides, Echinochloa oryzicola Vasing. ), Cyperus difformis L. , Monochoria vaginalis ( Burm. f. ) Presl. var. plantaginea ( Roxb. ) Solms-Laub.), annual weeds such as batttuk oepul (Lindernia pyxidara L.) (annual weed ), and beech bangdongsani (Cyperus serotinus Rottb.), olbanggae (Eleocharis kuroguwai Ohwi), the ladle goraeng (Scirpus juncoides Roxb. subsp Perennial weeds, such as hotarui ( Ohwi ) T. Koyama ), can also be controlled with low doses over a wide range from pre-emergence to growth.

Hereinafter, the present invention will be described in more detail by way of Examples, but the technical scope of the present invention is not limited to these Examples.

In this example, glutathione conjugation activity against VLCFAE inhibitory herbicides was analyzed for GST designated as registration number: Q8GTC0 (referred to as "TaQ8GTC0" in this example) among GST of wheat.

<Construction of E. coli expression form of TaQ8GTC0 protein>

1 shows the configuration of constructing an E. coli expression form of the TaQ8GTC0 protein. First, cDNA was synthesized by reverse transcription reaction using RNA prepared from shoots of wheat (Agricultural Forestry and Forestry No. 61) as a template. PCR reaction was performed using the obtained cDNA as a template using a primer set TaQ8GTC0 to H (SEQ ID NO: 3) and TaQ8GTC0 to D (SEQ ID NO: 4) having an NdeI recognition site at the 5' end and a BamHI recognition site at the 3' end A TaQ8GTC0 gene fragment was obtained.

Then, the obtained TaQ8GTC0 gene fragment was treated with NdeI and BamHI, and the TaQ8GTC0 gene fragment was used as an insert, and pET22b(+), similarly treated with NdeI and BamHI, was used as a vector. Ligation was performed on these inserts and vectors. After the ligation reaction, the vector contained in the reaction solution was introduced into the E. coli JM109 strain using the reaction solution. Then, a plasmid was prepared from the colony in which the introduction of the target plasmid was confirmed by colony PCR. By sequencing, the nucleotide sequence of the TaQ8GTC0 gene present between the NdeI and BamHI cleavage sites was checked for errors by PCR. The obtained plasmid was introduced into an Escherichia coli BL21 (DE3) strain to construct an Escherichia coli expression body (KLB-606) expressing the TaQ8GTC0 protein.

<Production of TaQ8GTC0 protein>

TaQ8GTC0 protein was expressed using the constructed E. coli BL21 (DE3) strain (KLB-606). A single colony was inoculated into an LB broth containing 50 ppm ampicillin, and a solution cultured in a test tube overnight was used as a preculture solution. 2.5ml of the pre-culture solution was added to 250ml of 50ppm ampicillin-containing LB liquid medium (1L Erlenmeyer flask), and _{cultured at 37°C and 200rpm until OD 600} = 0.5-0.6. Then, after cooling on ice for 5 minutes, IPTG was added to a final concentration of 1 mM, and TaQ8GTC0 protein was induced for 21 hours at 27 ° C. and 200 rpm. Then, cells were collected (centrifuged at 4 ° C, 6000 x g for 10 minutes). The cells were stored at -80°C. 30ml of PBS buffer (0.14M NaCl, 2.7mM KCl, 10mM Na ₂ HPO ₄ , 1.8mM KH ₂ PO ₄ , pH 7.3) was added to cryopreserved cells (0.5L culture) and sonicated (TAITEC) VP-30S microchip, output 3, 15 sec constant x 7 to 8 times), and then centrifuged for 20 minutes at 4°C and 15,000 x g to obtain a supernatant. The coenzyme solution was loaded onto a GSTrap 4B column (bed vol. 1ml) equilibrated with PBS buffer at a flow rate of 1ml/min, and the glutathione affinity column was washed with 20ml or more of PBS buffer, and then 2mL of elution buffer (50mM). Tris-HCl, 10 mM reduced glutathione, pH 8.0) was injected into the column to elute the TaQ8GTC0 protein ( FIG. 2 ). This protein solution was substituted with PBS buffer by ultrafiltration using Nanosep (10K), and stored at -80°C. The protein concentration was measured by the Bradford method according to the TaKaRa Bradford Protein Assay Kit (Takara) manual. In Figure 2, M is a molecular weight marker. In the results of FIG. 2 , 8.0 μg of total protein was added to the lanes of the crude enzyme and the pass-through fraction. In FIG. 2 , the coenzyme is a supernatant obtained by centrifuging the cells by sonication, and the flow-through fraction is a solution obtained by loading the crude enzyme solution into an affinity column.

<Analysis of glutathione conjugation activity against VLCFAE inhibitory herbicides>

All VLCFAE inhibitory herbicides used in this Example were added to the reaction solution in the form of an acetone solution. The conjugation reaction for the VLCFAE inhibitory herbicide was 50 µl of 100 mM potassium phosphate buffer (pH 6.8), 10 µl of 1 mM VLCFAE inhibitory herbicide, 20 µl of 10 mM reduced glutathione (pH 7.0), 6 µl of TaQ8GTC0 protein (total 378ng, PBS buffer) and 114 μl of PBS buffer were made into a total of 200 μl, reacted at 30° C. for 15 minutes, and then 50 μl of the filtered solution using a 0.2 μm filter was injected into HPLC. The HPLC analysis conditions are shown below.

Device: Agilent 1100 series

Column: CAPCELL PAK C18 AQ 4.6mmI.D.x250mm (SHISEIDO)

Mobile phase: acetonitrile/water = 5/95 (hold for 5 minutes) → (4 minutes) → 40/60 (hold for 4 minutes) → (4 minutes) → 90/10 (hold for 8 minutes) → (1 minute) → 5 /95 (hold for 4 minutes) (each solvent contains 0.5% acetic acid)

Temperature: 35℃

Flow rate: 1.0ml/min

Detection: 254nm

<Results and considerations>

The VLCFAE inhibitory herbicide tested for the glutathione-conjugation activity of the TaQ8GTC0 protein is shown in FIG. 3 . When analyzing the glutathione-conjugation activity for each VLCFAE inhibitory herbicide, the production amount of glutathione conjugate (GS conjugate) was measured as the decrease amount of the VLCFAE inhibitory herbicide in the reaction solution. The glutathione-conjugation activity of the TaQ8GTC0 protein for each VLCFAE inhibitory herbicide is shown in Table 1 below.

herbicide type Enzyme activity (mmol/mg protein/15 min) ^a) Pyroxasulfone Isoxazoline 11.7 ± 3.7 Fenoxasulfone 19.5 ± 8.4 Metolachlor Chloroacetanilide 2.2 ± 0.8 Alachlor 1.5 ± 0.1 Flufenacet Oxyacetamide nd ^b) Mefenacet n.d. Anilofos Dithiophosphoric acid n.d. Piperophos n.d. Fentrazamide Tetrazolinone 0.93 ± 0.2 Cafenstrole Triazole 0.46 ± 0.1 Indanofan Oxirane nd ^b)

a) Enzyme activity was expressed as the mean ± standard deviation of 7 experiments for pyroxasulfone and 4 experiments for other drugs. b) not detected

As an example, the HPLC chromatogram obtained when pyroxasulfone and glutathione were conjugated and pyroxasulfone was used as a VLCFAE inhibitory herbicide is shown in FIG. 4 . In the HPLC chromatogram shown in FIG. 4 , the upper part is the glutathione binding activity test result containing the TaQ8GTC0 protein, the middle part is the glutathione binding activity test result without the TaQ8GTC0 protein, and the bottom part is the glutathione binding activity test result that does not include the TaQ8GTC0 protein and glutathione. Each result is shown.

As shown in Table 1, the TaQ8GTC0 protein was found to have a specifically high conjugation activity against pyroxasulfone and phenoxasulfone having an isoxazoline backbone among VLCFAE inhibitory herbicides. On the other hand, in the case of a VLCFAE inhibitory herbicide having a structure other than the isoxazoline backbone, it was found that the glutathione-conjugation activity by the TaQ8GTC0 protein was significantly low. In addition, as shown in FIG. 4 , in the reaction solution using pyroxasulfone as a substrate, a peak of the GS conjugate of pyroxasulfone (M-15) was confirmed. This indicates that 4.81 nmol of the total amount of 10 nmol of pyroxasulfone added to the reaction solution became a GS conjugate.

In this Example, the conjugation activity for carpenstrole was measured by quantifying the compound released by glutathione conjugation of this herbicide. In addition, since the peaks of the glutathione conjugates of the eight herbicides (metolaclo, alaclo, flufenacet, mefenacet, pentolazamide, indanophane, anilophos, and piperophos) were unclear, When analyzing the glutathione-conjugation activity, first change the reaction conditions (increase the amount of enzyme added to the reaction solution or lengthen the reaction time), generate a glutathione conjugate (GS conjugate), and identify the peak by LC/MS.

In this example, transgenic rice into which the TaQ8GTC0 gene was introduced was constructed, and the sensitivity of the transgenic rice to VLCFAE inhibitory herbicides was tested.

<Construction of transgenic rice>

In this example, as shown in FIG. 5 , a vector (R-5-TaQ8GTC0) for rice transformation of the TaQ8GTC0 gene was constructed.

Specifically, cDNA was synthesized by reverse transcription reaction using RNA prepared from the leaves of wheat (Nonglim No. 61) as a template. Using the obtained cDNA as a template, PCR reaction was performed using a primer set of TaQ8GTC0 to M (SEQ ID NO: 5) and TaQ8GTC0 to N (SEQ ID NO: 6), and a SalI recognition site at the 5' end and a NotI recognition site at the 3' end A gene fragment of the TaQ8GTC0 gene with

Then, the obtained TaQ8GTC0 gene fragment was treated with SalI and NotI, and then the TaQ8GTC0 gene fragment was used as an insert, and pENTR-1A (Thermo Fisher Scientific Inc.) treated similarly with SalI and NotI was used as a vector. A binding reaction was performed using these inserts and vectors. The thus constructed entry clone (pENTR1A-TaQ8GTC0) was transformed and introduced into Escherichia coli JM109 strain. Then, a plasmid was prepared from a colony in which introduction of the target plasmid was confirmed by colony PCR. By sequencing, the nucleotide sequence of the TaQ8GTC0 gene present between the SalI and NotI cleavage sites was checked for errors by PCR.

Next, the constructed entry clone (pENTR1A-TaQ8GTC0) (KLB-649) and the target vector (destination vector), PalSelect R-5 (Inplanta Innovations Inc.), were subjected to LR reaction and the constructed expression vector (PalSelect R-5 A vector for rice transformation in which the TaQ8GTC0 gene was inserted between the attB sequences) was introduced into E. coli HST02 strain by transformation. Then, a plasmid was prepared from a colony in which the introduction of the target plasmid was confirmed by colony PCR, and the nucleotide sequence of the TaQ8GTC0 gene inserted between the attB sequences was confirmed by sequencing analysis (KLB-650).

<Introduction of TaQ8GTC0 gene into rice by transformation>

The constructed rice transformation vector KLB-650 (PalSelect R-5-TaQ8GTC0) was introduced into Agrobacterium (EHA105) by electroporation (KLB-654). Then, the TaQ8GTC0 gene was introduced into the cultured cells of rice by the Agrobacterium method (S. Toki Plant Mol. Biol. Rep. 15 16-21 (1997)), and then selected with bispiribac sodium salt (BS). .

The TaQ8GTC0 gene was transformed into rice and cultured cells introduced with the TaQ8GTC0 gene were selected with 0.25 μM bispyrivac sodium salt (BS) for 1 month. 279 (positive control in which the GFP gene was introduced instead of the TaQ8GTC0 gene) was confirmed as in (FIG. 6). After transplanting the cultured cells of this transgenic rice into a drug-free redifferentiation medium, the obtained plants were sequentially cultivated in an isolation greenhouse. Ear emergence was confirmed in all plants on average 2 months from the start of cultivation in the quarantine greenhouse, and progeny seeds (T1) were harvested from these plants.

<TaQ8GTC0 transgenic rice pyroxasulfone resistance>

For 18 lineages of rice plants, resistance to pyroxasulfone was investigated in a germination inhibition test using gellan gum as a medium.

Hoagland's mix and 3 g of gellan gum were suspended in 1 L of distilled water, and then sufficiently dissolved in a microwave oven warmly. 15 ml of it was injected into a tubular bottle before cooling (30 ml injected into the plate). The gellan gum medium was injected into a tubular bottle or plate, and the drug was added and mixed well. Rice hull was immersed in a 50-fold diluted sodium hypochlorite solution (antiformin, Wako) for about 20 minutes and then washed well. Sterilized seeds were soaked in distilled water and left at 27°C until germination (about 2 days). The germinated seeds were lightly transplanted into the gellan gum medium (plate) containing 0.25 μM BS with the sprout facing up. These and a beaker containing distilled water were put together in a transparent case, covered with a wrapper, and grown for 2 days at 27° C. under fluorescent lighting (light 14 hours, dark 10 hours). Then, a gellan containing ^{pyroxasulfone (final concentration 10 -8} , 10 ^-7 , 10 ^-6 , 10 ^-5 M) of rice seeds judged to have BS resistance based on root elongation as an index Transplanted into gum medium (tubular bottle). Then, put them together in a beaker with distilled water in a transparent case, cover the lid with a wrap, and grow at 27°C under fluorescent lighting (light 14 hours, dark 10 hours) for 4 to 5 days, and then measure the plant height. did Inhibition of the drug was calculated by using the plant height as an index to calculate the growth inhibition rate for the group without the drug, and the 50% growth inhibition concentration (IC ₅₀ value) was measured by the Probit method.

Table 2 below shows the results of the above-described TaQ8GTC0 transgenic rice tolerance test against pyroxasulfone.

system IC ₅₀ (nM) Pyroxasulfone resistance ¹⁾ Wild 39 - #1-3 1139 29 #1-4 1306 33 #1-7 969 25 #1-10 510 13 #2-6 968 25 #2-9 1091 28 #3-4 1720 44 #3-9 1007 26 #3-12 67 2 #3-13 13751 353 #4-1 496 13 #4-3 4315 111 #4-7 7146 183 #4-10 2118 54 #4-13 3852 99 #4-14 45 One #5-1 968 25 #5-11 983 25

1) in Table 2 _{is the value of IC 50} (transgenic rice)/IC ₅₀ (wild type rice).

As shown in Table 2, 16 strains out of 18 strains used in the test showed more than 10-fold resistance to pyroxasulfone compared to the wild-type, and the strongest strain (#3-13) showed about 350-fold resistance. .

<Determination of the minimum concentration of VLCFAE inhibitory herbicide that inhibits the growth of wild-type rice>

Since the drug tolerance of the TaQ8GTC0 transgenic rice is judged by the difference in sensitivity from wild-type rice as an indicator, the sensitivity test of wild-type rice (Nipponbare) to the VLCFAE inhibitory herbicide subject to the glutathione conjugation activity test was performed as follows.

A germination inhibition test was performed using gellan gum as a medium. 3 g of Hogrand Mix gellan gum was suspended in 1 L of distilled water, and then heated in a microwave to fully dissolve. Of this, 15 ml was injected into a tubular bottle before cooling. When the drug (acetone solution) was injected into the tubular bottle, it was added at the same time and mixed well (final acetone concentration was 0.1%). Rice bran was immersed in a 50-fold diluted sodium hypochlorite solution (antiformin, Wako) for about 20 minutes and then washed well. The sterilized seeds were soaked in distilled water and left at a temperature of 27°C until germination (about 2 days). The germinated seeds were transplanted into the drug-containing gellan gum medium (tubular bottle) with the sprout side up. Then, these and a beaker containing distilled water were put together in a transparent case, covered with a lid, and grown at 27° C. under fluorescent lighting (light 14 hours, dark 10 hours) for 1 week, and then the plants were sampled and the plant height was measured. Inhibition of the drug was calculated by using the plant height as an index to calculate the growth inhibition rate for the group without the drug, and the 50% growth inhibition concentration (IC ₅₀ value) was measured by the Probit method.

The sensitivity of wild-type rice (Nipponbare) to the VLCFAE inhibitory herbicide tested for glutathione-conjugation activity was tested. _{The IC 50} values for the growth of wild-type rice and the minimum concentration inhibiting the growth of each drug revealed from the test results are shown in Table 3 below.

drug IC ₅₀ (nM) Minimum inhibitory concentration (mM) Pyroxasulfone 48 0.075 Fenoxasulfone 54 0.05 Flufenacet 42 0.075 Mefenacet 388 0.75 Alachlor 332 0.5 Metolachlor 636 0.75 Anilofos 377 0.5 Piperophos 1311 2.5 Fentrazamide 46 0.5 Indanofan 61 0.25 Cafenstrole 131 0.75

<TaQ8GTC0 transgenic rice tolerance test to VLCFAE inhibitory herbicides>

The tolerance test to the VLCFAE inhibitory herbicide of the TaQ8GTC0 transgenic rice was conducted in the same manner as the pyroxasulfone tolerance test described above. In this test example, as shown in Table 4, a total of 3 of the minimum concentration that completely inhibits the growth of wild-type rice (Nipponbare) (x1), the concentration 4 times the minimum concentration (x4), and the concentration 16 times the minimum concentration (x16) A tolerance test to VLCFAE inhibitory herbicides was conducted at dog concentrations.

drug Test Concentration (mM) x 1 ^a) x 4 ^b) x 16 ^c) Pyroxasulfone 0.075 0.30 1.2 Fenoxasulfone 0.05 0.20 0.8 Flufenacet 0.075 0.30 1.2 Mefenacet 0.75 3.0 12 Alachlor 0.5 2.0 8.0 Metolachlor 0.75 3.0 12 Anilofos 0.5 2.0 8.0 Piperophos 2.5 10 40 Fentrazamide 0.5 2.0 8.0 Indanofan 0.25 1.0 4.0 Cafenstrole 0.75 3.0 12

In Table 4, a) shows the minimum concentration inhibiting the growth of wild-type rice (Nipponbare), b) shows the 4-fold concentration of a), and c) shows the 16-fold concentration of a).

In this test example, using wild-type rice as a comparative control, the #3-13 line TaQ8GTC0 transgenic rice showing strong resistance to pyroxasulfone was used. The results are shown in Figures 7a to 7f. In addition, in FIGS. 7a to 7f, going from left to right, the drug concentration 0 μM, the minimum concentration of Table 4 (x1), the 4-fold concentration of the minimum concentration of Table 4 (x4), the minimum concentration of 16 times the concentration of Table 4 (x16) indicates

In the group treated with pyroxasulfone and phenoxasulfone, even at the highest concentration of the test (16 times the minimum concentration), the TaQ8GTC0 gene transduced rice showed strong resistance without showing an effect (Fig. 7a), whereas the six drugs ( In the treatment groups of flufenacet, anilophos, piperophos, carpenstrol, indanofan, and fantrazamide), growth was inhibited even at the minimum concentration, and the TaQ8GTC0 transgenic rice showed no resistance to these drugs. (FIG. 7B-FIG. 7D). In addition, for the three drugs (metolaclo, alaclo, mefenacet), growth was not affected at the minimum concentration, but growth was inhibited at a concentration four times the minimum concentration (x4). The tolerance of the TaQ8GTC0 transgenic rice was judged to be very weak, which did not reach a practical level ( FIGS. 7e and 7f ).

In addition, although data are not presented, the sensitivity intensity to various drugs of the #4-7 line, which showed a strong resistance on par with the #3-13 line, to pyroxasulfone was the same as that of #3-13.

As described above, it was found that the TaQ8GTC0 transgenic rice exhibited specific resistance to isoxazoline-based herbicides among VLCFAE inhibitory herbicides. In addition, the drug resistance of the present transgenic rice was largely correlated with the glutathione-conjugation activity of the TaQ8GTC0 protein.

In this example, transgenic Arabidopsis thaliana introduced with the TaQ8GTC0 gene was constructed, and the transgenic Arabidopsis thaliana was tested for sensitivity to VLCFAE inhibitory herbicides.

<Construction of transgenic Arabidopsis>

First, as shown in FIG. 8, an Arabidopsis transformation vector (A-3-TaQ8GTC0) having a TaQ8GTC0 gene was constructed. Specifically, the entry clone (pENTR1A-TaQ8GTC0, KLB-649) in which the TaQ8GTC0 gene (ORF) was inserted between attL1 and attL2 of pENTR1A (Thermo Fisher Scientific Inc.) and PalSelect A-3 (Inplanta), a destination vector Innovations Inc.) and LR reaction to construct a vector for transformation of Arabidopsis thaliana. This vector for transformation of Arabidopsis is obtained by inserting the TaQ8GTC0 gene between the attB sequence of PalSelect A-3. Next, the constructed Arabidopsis transformation vector was introduced into E. coli HST02 strain by transformation. Then, a plasmid was prepared from the colony in which the introduction of the target plasmid was confirmed by colony PCR, and it was confirmed by sequencing that the nucleotide sequence of the TaQ8GTC0 gene inserted between the attB sequences was correct (KLB-707).

<Introduction of TaQ8GTC0 gene into Arabidopsis by transformation>

The Arabidopsis transformation vector (PalSelect A-3-TaQ8GTC0) constructed as described above was introduced into Agrobacterium (EHA105) by electroporation (KLB-718). Next, the TaQ8GTC0 gene was introduced into Arabidopsis thaliana by the floral dip method (S.J. Clough et al., Plant J. 16 735-743 (1998)), and then selected with bispyrivac sodium salt (BS).

<Identification of transgenic Arabidopsis genes>

A simple method using Ampdirect Plus sample solution (Shimadzu) [20 mM Tris-HCl (pH 8.0), 5 mM EDTA, 400 mM NaCl, 0.3% SDS, 200 μg/ml Proteinase K] in the transgenic Arabidopsis plant obtained as described above genomic DNA was extracted. Using this as a template, KAPA 3G DNA polymerase (KAPA BIOSYSTEMS), the introduction of the gene was confirmed by PCR under the following conditions.

PCR was performed in a total of 50 μl of reaction solution (0.4 μl template, 0.3 μl 50 μM sense primer (Sequence(TaQ8GTC0)-2: SEQ ID NO: 7), 0.3 μl 50 μM antisense primer (NOSter-13: SEQ ID NO: 8), 25 μl KAPA Plant) PCR buffer, 0.4 μl KAPA 3G DNA polymerase, 23.6 μl sterile water). PCR was performed with initial transformation: 20 s at 95 °C, transformation: 20 s at 95 °C, ligation: 15 s at 58 °C, extension: 30 s at 72 °C (40 cycles), final extension: 4 min at 72 °C .

Next, the TaQ8GTC0 gene was introduced into Arabidopsis by transformation using Agrobacterium (KLB-718) introduced with an Arabidopsis transformation vector (PalSelect A-3-TaQ8GTC0). Then, the seeds (approximately 50,000 in total) collected from the transformed Arabidopsis plants were sown in a selection medium containing 0.1 μM BS, and the presence or absence of a selection marker (W574L/S653I mutant Arabidopsis ALS) was used as an indicator to transform transformants. was selected. As a result, it was confirmed that a total of 30 individuals were growing (FIG. 9). The photograph shown in FIG. 9 shows the appearance 14 days after sowing, and shows 3 out of a total of 30 transformants grown in the selection medium. In addition, as a result of selecting 10 of them and performing PCR reaction using genomic DNA simply extracted from these leaves as a template, introduction of the target wheat GST gene was confirmed (FIG. 10). In FIG. 10, lanes 1 to 10 show PCR results using genomic DNA extracted from each individual as a template by selecting 10 individuals from a total of 30 individuals grown in the selection medium. Region A (563bp) was amplified by PCR using the primer sets of Sequence(TaQ8GTC0)-2 and Noster-13 described above. In the electrophoresis picture of FIG. 10 , the left lane is a 100 bp DNA ladder.

<TaQ8GTC0 gene-introduced Arabidopsis pyroxasulfone tolerance test>

In order to examine the resistance to pyroxasulfone of Arabidopsis thaliana introduced with the TaQ8GTC0 gene, a medium was prepared as follows: First, Murashige-Skoog (MS) medium (1 bag), thiamine hydrochloride (3 mg) , nicotinic acid (5 mg), pyridoxine hydrochloride (0.5 mg), and sucrose (10 g) were added to a 1 L beaker, then the pH was adjusted to 5.7, the volume was adjusted to 1 L, and 8 g (0.8%) of agar was added. This was autoclaved, cooled to room temperature, and pyroxasulfone (acetone solution) was added thereto. A 30ml fraction thereof was dispensed twice on each plate (No. 2 square plate), and MS medium containing pyroxasulfone of a predetermined concentration was prepared.

Using this medium, the pyroxasulfone sensitivity test of transgenic Arabidopsis thaliana was performed as follows: dry seeds of Arabidopsis thaliana in the required amount for 2 minutes in 70% ethanol, 15 in hypochlorous acid (0.02% Triton-X-100) It was stirred for minutes and washed 10 times with sterile water. Then, the seeds were suspended in 1 ml of an autoclaved 0.1% agar solution, and the solution was mixed well to make it uniform, and then a total of 30 seeds were dented on the surface of the medium one by one. It was sealed with a surgical tape, left at 4°C for 2 days, and then transferred to 22°C to germinate.

The test results for wild-type Arabidopsis thaliana (Columbia-0) are shown in FIG. 11, and the test results for Arabidopsis thaliana introduced with the TaQ8GTC0 gene are shown in FIG. In addition, the photos shown in FIGS. 11 and 12 are photos taken after growing at 22° C. for 14 days.

As shown in FIG. 11, wild-type Arabidopsis thaliana was somewhat inhibited at the concentration of pyroxasulfone at 100 nM, and almost completely inhibited at 1 μM or higher. In contrast, as shown in FIG. 12, Arabidopsis thaliana introduced with the TaQ8GTC0 gene not only grows at the pyroxasulfone concentration of 1 μM in the same way as in the control without the drug, but also has some growth inhibition at 10 μM compared to 1 μM, but It was found that it was sufficiently grown and exhibited strong resistance to pyroxasulfone. In this test example, Arabidopsis thaliana introduced with the TaQ8GTC0 gene of the tested 7 strains exhibited at least 10-fold resistance to pyroxasulfone compared to the wild type (Columbia-0). In addition, Figure 12 shows the results of transgenic Arabidopsis thaliana (KLB-718 1-1-5), which exhibits particularly strong resistance. From these results, it was found that wheat GST (TaQ8GTC0) functions in Arabidopsis and confers resistance to pyroxasulfone.

<Sensibility test of Arabidopsis plants to VLCFAE inhibitory herbicides>

In this test example, the sensitivity test of wild-type Arabidopsis thaliana to VLCFAE inhibitory herbicides was performed in the same manner as the pyroxasulfone tolerance test of Arabidopsis thaliana into which the TaQ8GTC0 gene was introduced.

First, Table 5 shows the results of determining the concentration of the VLCFAE inhibitory herbicide that strongly inhibits the growth of wild-type Arabidopsis.

drug Test Concentration (mM) Pyroxasulfone One Fenoxasulfone One Flufenacet One Mefenacet 100 Alachlor 100 Metolachlor 100 Anilofos 10 Piperophos 100 Fentrazamide 10 Indanofan 100 Cafenstrole One

Next, the drug sensitivity of Arabidopsis thaliana introduced with the TaQ8GTC0 gene to VLCFAE inhibitory herbicides was investigated by employing these test concentrations, and the results are shown in FIGS. 13 and 14 . The pictures shown in FIGS. 13 and 14 are pictures taken after being grown for 14 days at a temperature of 22°C. 13 and 14, in Arabidopsis thaliana introduced with the TaQ8GTC0 gene (test KLB-718 1-1-5, which has the strongest resistance), isoxazoline-based pyroxasulfone and fenoxasulfone ) showed strong resistance to (FIG. 13). On the other hand, for the other 9 types of drugs, the growth of Arabidopsis thaliana into which the TaQ8GTC0 gene was introduced was strongly inhibited as in the wild type ( FIGS. 13 and 14 ).

From the above results, Arabidopsis thaliana introduced with the TaQ8GTC0 gene was found to exhibit specific resistance to isoxazoline-based herbicides. In addition, drug resistance of Arabidopsis thaliana introduced with TaQ8GTC0 gene was largely correlated with glutathione-conjugation activity of TaQ8GTC0 protein.

In this example, the resistance to high temperature stress of transgenic rice introduced with the TaQ8GTC0 gene was tested.

First, in the same manner as the pyroxasulfone tolerance test of rice in which the TaQ8GTC0 gene was introduced, the TaQ8GTC0 gene introduced rice seeds judged to have BS resistance by elongation of the root part of the gellan gum medium (plate) were transplanted into a plastic cup. (For wild-type rice, individuals grown in BS-free gellan gum medium were transplanted into plastic cups). After being grown for 1 week in an isolation greenhouse, high-temperature treatment (50° C., 2 hours 30 minutes), and grown for 1 week in an isolation greenhouse, the plant height and root weight of the rice were measured. In addition, in this Example, #4-7 and #3-13 of the TaQ8GTC0 transgenic rice prepared in Example 2 were used.

Since similar results were obtained in a total of three tests, the results of one test among them are shown in FIG. 15 . As a result of the test, it was found that the TaQ8GTC0 transgenic rice was less affected by high temperature treatment than the wild-type rice in both plant length and root weight, and that the prepared TaQ8GTC0 transgenic rice had heat stress tolerance.

Comparative Example 1

In this comparative example, the metabolic activity of pyroxazoline was analyzed with respect to GST of other plants having high homology with TaQ8GTC0, which is involved in resistance to isoxazoline derivatives and resistance to high temperature stress in Examples 1 to 4.

Specifically, in FIG. 16, 11 molecular species of wheat and corn-derived GST having high homology with TaQ8GTC0 and the TaQ8GTC0, and 3 molecular species of rice-derived GST having the same functional metolaclo metabolic activity (I. Cummins et al., Plant Mol. Biol. 52, 591-603 (2003); B. McGonigle et al., Plant Physiol. 124, 1105-1120 (2000); HY Cho et al. Pestic. Biochem. Physiol. 83, 29-36 (2005) ; HY Cho et al. Pestic. Biochem. Physiol. 86, 110-115 (2006); and HY Cho et al. J. Biochem. Mol. Biol. 40, 511-516 (2007)) shows the GST of a total of 14 plants. . In this comparative example, rice cultured cells into which each of these plant GST genes were introduced were prepared, and the presence or absence of resistance to pyroxasulfone was confirmed. In addition, numbers in parentheses in FIG. 16 indicate homology to the amino acid sequence of wheat GST (TaQ8GTC0).

Specifically, for gene 7, the presence or absence of metabolic activity was analyzed using the fatty acid content of cultured rice cells cultured in liquid culture in a medium containing pyroxasulfonic acid and the proliferation of rice cultured cells on a drug-containing plate for gene 2 as an indicator.

<Analysis of pyroxasulfone metabolic activity using fatty acid content as an index in cultured rice cells>

First, as shown in FIG. 17 , a vector for rice transformation was constructed. In this example, cDNA was prepared by reverse transcription reaction of RNA prepared from leaves of rice (Nipponbare), wheat (Agricultural & Forestry No. 61) and corn (Pioneer 32K61). Using the obtained cDNA as a template, the full length of each GST gene with XbaI at the 5' end and AflII restriction enzyme sites at the 3' end was amplified by PCR. Then, the 3' end was treated with AflII _{, blunted with T 4} DNA enzyme, and then the 5' end was treated with restriction enzyme XbaI. Then, after SacI treatment with KLB-224 in which the CSP (rice callus-specific promoter: Gene Locus Os10g0207500)::GUS::NOSt construct was introduced into the MCS region of PalSelect R-4 (Inplanta Innovations Inc), T ₄ A vector fragment was obtained by blunting with a DNA enzyme and XbaI treatment.

The vector constructed by combining the above GST gene fragment and the vector fragment was introduced into Escherichia coli (HST-02 strain) by transformation. A part of the transformation reaction solution was applied to a 50 ppm spectinomycin-containing LB solid medium and cultured overnight at 37° C., and colonies into which the target vector was introduced were selected by PCR using several grown colonies as templates. The colonies were inoculated in YM broth containing 50 ppm spectinomycin and cultured overnight at 37° C., and then a plasmid was prepared from the cells. Then, the entire sequence of the target gene inserted into the vector and the nucleotide sequences of both ends of 5' and 3' were analyzed through sequencing. The nucleotide sequences of the primers used in constructing the vector for rice transformation into which each GST gene is inserted are shown below.

The following primer sets were used for OsGSTF5 derived from rice.

OsGSTF5-1: 5'-AAAAAATCTAGAAAAGTGCAGGGCAAATTC-3' (sense: SEQ ID NO: 9)

OsGSTF5-2: 5'-AAAAAACTTAAGCTATGGTATGTTCCCACT-3' (antisense: SEQ ID NO: 10)

The following primer sets were used for OsGSTU5 derived from rice.

OsGSTU5-1: 5'-AAAAAATCTAGAATCTTCTTCTCCGACGAG-3' (sense: SEQ ID NO: 11)

OsGSTU5-2: 5'-AAAAAACTTAAGCTACTTGGCGCCAAACTT-3' (antisense: SEQ ID NO: 12)

The following primer sets were used for wheat-derived TaQ8GTB9.

TaQ8GTB9(GSTF4)-1: 5'-AAAAAATCTAGAATGGAGCCTATGAAGGTG-3' (sense: SEQ ID NO: 13)

TaQ8GTB9(GSTF4)-2: 5'-AAAAAACTTAAGTCATGGTATTCTCCCGCT-3' (antisense: SEQ ID NO: 14)

The following primer sets were used for ZmB6T8R4 from maize.

ZmB6T8R4(Corn)-3: 5'-AAAAAATCTAGATCGTTTCGAGGCCGAT-3' (sense: SEQ ID NO: 15)

ZmB6T8R4(Corn)-2: 5'-AAAAAACTTAAGTCACTTGGCCCCGAACTT-3' (antisense: SEQ ID NO: 16)

The following primer sets were used for ZmQ9ZP61 derived from corn.

ZmQ9ZP61(GST6)-3: 5'-AAAAAATCTAGATACAGCCACGTCGCTT-3' (sense: SEQ ID NO: 17)

ZmQ9ZP61(GST6)-2: 5'-AAAAAACTTAAGTCACTTGGCCCCGAACTT-3' (antisense: SEQ ID NO: 18)

The following primer sets were used for ZmM16901 from corn.

M16901(GSTI)-3: 5'-AAAAAATCTAGAGTTGGGTCTGGGACAC-3' (sense: SEQ ID NO: 19)

M16901(GSTI)-2: 5'-AAAAAACTTAAGTCAAGCAGATGGCTTCAT-3' (antisense: SEQ ID NO: 20)

The following primer sets were used for ZmY12862 from corn.

ZmY12862(GST5)-1: 5'-AAAAAATCTAGAATGGCCGAGGAGAAGAAG-3' (sense: SEQ ID NO: 21)

ZmY12862(GST5)-2: 5'-AAAAAACTTAAGCTACTCGATGCCCAGCCT-3' (antisense: SEQ ID NO: 22)

That is, a vector for rice transformation (PalSelect R-4-GST) was constructed for each of 7 genes out of a total of 14 genes shown in FIG. 16 . In addition, for some genes, the sequence inserted into the vector differed from the sequence in the database. These differences are shown in Table 6.

GST gene base sequence difference difference in amino acid sequence TaQ8GTB9 C->G, position 84 Asp(D)->Glu(E) T->G, position 178 Tyr(Y)->Asp(D) C->G, position 315 His(H)->Gln(Q) ZmM16901 C->A, position 207 - G->T, position 363 - T->C, position 528 - ZmB6T8R4 A->G, position 532 Ile(I)->Val(V) G->C, position 565 Ala(A)->Pro(P) G->C, position 603 - C->A, position 604 Leu(L)->Met(M) G->A, position 629 Gly(G)->Glu(E) ZmY12862 A->C, position 79 Met(M)->Leu(L) G->A, position 124 Gly(G)->Arg(R) C->A, position 251 Ala(A)->Glu(E) C->G, position 333 - C->T, position 453 -

In Table 6, "-" means that there is no difference in the amino acid sequence. In the case of OsGSTF5, OsGSTU5, and ZmQ9ZP61, the sequences in the database were completely matched.

<Introduction of GST gene into rice by transformation>

The vector for rice transformation (PalSelect R-4-GST) having various GST genes constructed as described above was introduced into Agrobacterium (EHA105) by electroporation. Then, the GST gene was introduced into rice cultured cells according to the Agrobacterium method (S. Toki Plant Mol. Biol. Rep. 15 16-21 (1997)), and then, bispiribac sodium salt (BS) was selected.

<Cultivation of transformed rice cells>

The transformed rice cells constructed as described above were cultured in the presence of pyroxasulfonic acid. Pyroxasulfone was added to the liquid medium as an acetone solution. The final acetone concentration was 0.1%. In culture, cultured cells of rice transformed with KLB-279 (plasmid containing Act1p (actin 1 promoter of rice): sGFP:: NOSt in the MCS portion of PalSelect R-4) were used as a control. It cultured by the following method.

1 bag of mixed salt (MS inorganic salt) for Murashige-Skoog medium, 1 ml of thiamine hydrochloride at 10 mg/ml, 1 ml of nicotinic acid at 5 mg/ml, 1 ml of pyridoxine hydrochloride at 10 mg/ml, 1 ml of glycine at 2 mg/ml , 0.2mg/ml 2,4-D 1ml, 30g sucrose (sucrose), 50mg/ml myo-inositol 1ml into a 1L beaker, adjust the pH to 5.7, make exactly 1000ml with distilled water, and then autoclave did After adding 0.05 ml of pyroxasulfone to a 200 ml Erlenmeyer flask containing 50 ml of this liquid medium, control rice cultured cells grown in a solid medium containing 3 g of gellan gum and 0.25 μM BS in the same composition as above, and rice prepared as above About 0.5 g of transformed cultured cells were added and cultured for 14 to 17 days. After culturing, the liquid medium was removed and rice cultured cells were recovered.

After transformation, cultured cells of rice into which the GST gene was introduced were selected in 0.25 μM bispiribac sodium salt (BS) for 1 month. Then, the cultured cells of fresh rice grown in the selection medium were mass-cultured in a conical beaker for about 1 month, and then cultured in a medium containing pyroxasulfonic acid. ^{The concentration of the drug during culture was 10 -7} M or 10 ^-6 M so that pyroxasulfone had an effect on fatty acid content in rice cultured cells (Y. Tanetani et al., Pestic. Biochem. Physiol. 95 47-55 (2009)) ). In addition, considering the possibility that mutant ALS may affect fatty acid content when functioning in rice cultured cells, KLB-279 (Act1p:: sGFP:: NOSt in the MCS portion of PalSelect R-4 included as a control) plasmid) transformed with rice cultured cells.

<Analysis of pyroxasulfone metabolic activity using fatty acid content as an index in cultured rice cells>

Fatty acids were extracted with undecenoic acid (C11:1) as an internal standard. Shoe containing C14:0, C16:0, C16:1, C18:0, C18:1, C18:2, C18:3, C20:0, C22:0, C22:1, C24:0 as analytical standards Each calibration curve was prepared and quantified using FAME mix of Supelco and other fatty acid methyl esters (C11:1, C15:0, C20:1, C26:0). Each fatty acid methyl ester was identified by the retention time and mass spectrum of gas chromatography (GC). In addition, quantitative and qualitative was performed with methyl ester, and in quantitative analysis, the amount of fatty acid was converted based on molecular weight.

For 1 g of cultured cells, in addition to 10 ml of water, 25 ml of methanol, and 12.5 ml of chloroform, the cultured cells were crushed using Hiscotron from Microtec Nition. At this time, 0.1 mg of an internal standard (10-undecenoic acid (C11:1)) dissolved in chloroform was added. The solution used for crushing is vacuum suction filtered, put into a separatory funnel, 40 ml of chloroform and 50 ml of saturated ammonium sulfate aqueous solution are added, and then left for about 30 minutes to sufficiently extract fatty acid lipids, and extraction of lipids with chloroform The operation was repeated 2 times (3 total times). The recovered chloroform layer was placed in a separatory funnel, washed with water, and the chloroform layer was recovered in an eggplant flask and concentrated in an evaporator. To a container dried under reduced pressure, 6 ml of 25% potassium hydroxide and 9 ml of ethanol were added, heated at 65° C. for 1 hour, and then 10% hydrochloric acid was added at room temperature to make it acidic (pH 2 or so). The solution was placed in a separatory funnel, 50 ml of hexane was added, stirred well, and the operation of recovering the organic layer was repeated twice, and then recovered by dehydration with solid anhydrous magnesium sulfate. After concentrating the hexane layer with an evaporator, it was dissolved in 2 ml of a mixed solvent (toluene/methanol = 4/1, v/v), and a few drops of concentrated sulfuric acid were added dropwise to the reaction solution, followed by reaction at 80° C. for 1 hour. After completion of the reaction, neutralized with saturated sodium bicarbonate water at room temperature was confirmed with a pH test paper, and the organic layer was recovered. 200 μl of the organic layer was injected into an Eppendorf tube with a filter, and 2 μl of the solution after centrifugation was analyzed by GC. The GC analysis conditions are shown below.

Device: Agilent 6890 Series GC System

Column: Supelco, Omegawax250 (30 m length, 0.25 mm inner diameter, 0.25 μm film thickness)

Injection temperature: 250℃

Initial temperature: 100℃

Initial time: 5min

Speed: 4℃/min

End temperature: 220℃

Last Time: 25min

Analyzer: FID

Analysis temperature: 250℃

From the analysis results, the fatty acid content in the cultured cells of 7 types of plant GST transgenic rice cultured in a medium containing pyroxasulfonic acid, the control rice cultured cells, and the control rice cultured cells not treated with the drug were shown in FIGS. 18 to 20. . The data displayed in Figs. 18 to 20 are all series of data. 18 to 20 show the results of analysis of fatty acids in cultured cells of two lines (line 1, line 2) rice for each GST gene.

In rice cultured cells cultured in a liquid medium containing pyroxasulfonic acid, the VLCFAE inhibitory action of pyroxasulfone significantly reduced the content of very long chain fatty acids (VLCFA), while the content of C15:0 (pentadecanoic acid) was significantly reduced. accumulated (Y. Tanetani et al., Pestic. Biochem. Physiol. 95, 47-55 (2009)). Based on the results of this study, the presence or absence of pyroxasulfone metabolic activity of each GST was determined. As a result, the C15:0 and VLCFA contents in the cultured cells of the rice cells into which each of the seven plant GST (TaQ8GTB9, ZmM16901, ZmB6T8R4, ZmQ9ZP61, ZmY12862, OsGSTF5, OsGSTU5) genes were introduced were the same as those of the control rice cells of all drug treatment groups. were the same, and it was judged that these GSTs did not have pyroxasulfone metabolic activity. In addition, the cultured cells of the two types of OsGST gene (F5, U5) transduced rice ^{were the results of the 10 -6} M treatment group, and there was no data from the cultured cells of the untreated control rice. All GST was judged to have no metabolic activity.

<Analysis of pyroxasulfone metabolic activity as an indicator of proliferation in the culture medium of rice cells>

Next, as shown in FIG. 21 , a vector for transforming rice having a maize GST gene was constructed. Also, in this test example, a vector for transformation of rice was constructed for each of the two types of maize GST genes (ZmQ9FQD1 and ZmB8A3K0), but the construction protocol is the same. described.

cDNA was synthesized by reverse transcription of RNA prepared from the foliage of corn (Pioneer 32K61). Using the synthesized cDNA as a template, a PCR reaction was performed with a primer set of ZmQ9FQD1-X and ZmQ9FQD1-Y to obtain a ZmQ9FQD1 gene fragment. Then, after the ZmQ9FQD1 gene fragment was treated with SalI and NotI, the ZmQ9FQD1 gene fragment was used as an insert, and pENTR-1A treated with SalI and NotI was used as a vector. A binding reaction was performed on these inserts and vectors. The thus constructed entry clone (pENTR1A-ZmQ9FQD1) was introduced into the E. coli JM109 strain. Then, a plasmid was prepared from a colony in which introduction of the target plasmid was confirmed by colony PCR. Through sequencing, it was confirmed that the nucleotide sequence of the inserted ZmQ9FQD1 gene was correct.

Then, an LR reaction was performed between the constructed entry clone (pENTR1A-ZmQ9FQD1) and the target vector, PalSelect R-5 (Inplanta Innovations Inc), to construct an expression vector (PalSelect R-5-ZmQ9FQD1). This expression vector (PalSelect R-5-ZmQ9FQD1) was introduced into E. coli HST02 strain by transformation. Then, a plasmid was prepared from a colony in which introduction of the target plasmid was confirmed by colony PCR. The nucleotide sequence of the inserted ZmQ9FQD1 gene was confirmed through sequencing. In addition, since the sequences of the two genes inserted into PalSelect R-5 were different from each other in the database, the differences are shown in Table 7.

GST gene base sequence difference difference in amino acid sequence ZmQ9FQD1 T->C, position 189 - G->C, position 381 Glu (E) -> Asp (D) T->C, position 476 Val(V)->Ala(A) G->C, position 477 - A->T, position 485 Asp(D)->Val(V) ZmB8A3K0 C->T, position 438 - A->G, position 532 Ile(I)->Val(V) G->C, position 567 - T->C, position 573 - G->C, position 603 - C->A, position 604 Leu(L)->Met(M)

In Table 7, "-" means no difference in amino acid sequence.

The primer sets used for PCR are shown below.

ZmB8A3K0-X: 5'-AAAAAAGTCGACATGGCGGCGGCGGCGGAG-3' (sense: SEQ ID NO:23)

ZmB8A3K0-Y: 5'-AAAAAAGCGGCCGCTCACTTGGCCCCGAACTTG-3' (antisense: SEQ ID NO: 24)

ZmQ9FQD1-X: 5'-AAAAAAGTCGACATGGCGCCGCCGATGAAG-3' (sense: SEQ ID NO:25)

ZmQ9FQD1-Y: 5'-AAAAAAGCGGCCGCCTATGGTATGTTCCCGCTG-3' (antisense: SEQ ID NO: 26)

<Introduction of GST gene into rice by transformation>

The vector for rice transformation (PalSelect R-5-ZmGST) for the two types of corn-derived GST genes constructed as described above was introduced into Agrobacterium (EHA105) by electroporation. Then, the TaQ8GTC0 gene was introduced into rice cultured cells by the Agrobacterium method (S. Toki Plant Mol. Biol. Rep. 15 16-21 (1997)), and then, bispyrivac sodium salt (BS) was used for selection.

Two types of corn GST genes were transformed and introduced into rice, respectively, and cultured cells of the rice cells into which the GST gene was introduced were selected with 0.25 μM bispiribac sodium salt (BS) for one month. As a result, as with KLB-279 (positive control in which the GFP gene was introduced instead of the TaQ8GTC0 gene), an individual actively proliferating in the selection medium was confirmed, suggesting that two lines of corn GST gene-transduced rice cultured cells could be constructed. was found (FIG. 22).

<Analysis of pyroxasulfone metabolic activity as an indicator of proliferation in cultured cell medium of rice>

The two cultured cells of the corn GST transgenic rice obtained as described above and the cultured cells of KLB-279 transgenic rice (comparative control) were plated in N6D medium, and 2 weeks later, the proliferation of each rice cultured cell was indexed. As a result, resistance to pyroxasulfone was investigated. Pyroxasulfone was tested with an acetone solution, and the final concentration of acetone added to the medium was 1%, and the final concentration of the drug was 10 ^-4 M.

The results are shown in FIG. 23 . The photograph of FIG. 23 shows the proliferation of transformed rice cells in a medium containing ^{100 μM (10 −4 M) of pyroxasulfonic acid.} As shown in FIG. 23 , 2 weeks after dentition, the GST gene rice cultured cells of the two lines were enlarged like the comparative control group. As such, the ZmB8A3K0 transgenic rice cultured cells and the ZmQ9FQD1 transgenic rice cultured cells did not proliferate like the comparative control and did not show resistance to pyroxasulfone. Therefore, it was determined from the results of this comparative example that GST derived from two types of corn did not have pyroxasulfone metabolic activity.

As mentioned above, from the results of this comparative example, it was found that rice GST, which is known to be involved in drug metabolism of a mechanism of action such as pyroxasulfonic acid, and wheat and corn GST, which are highly homologous to TaQ8GTC0, do not have pyroxasulfone metabolic activity. Therefore, it was found that the metabolic activity of TaQ8GTC0 to pyroxasulfone, shown in Examples 1 to 4, is specific that cannot be easily predicted from known information.

Comparative Example 2

In this comparative example, the glutathione conjugation activity was analyzed for GST (TaQ8GTC1) (cultivar: Hunter, Accession No.: AJ440791, homology 78%) of wheat having the highest amino acid homology to TaQ8GTC0.

<Clone of TaQ8GTC1 gene (Fig. 24)>

In this comparative example, 6 varieties of wheat (ie, Yumekaori, Hanamanten, Yumeseiki, Nohrin No. 61), Apache and Gatalina )) was used. TaQ8GTC1 gene in which an XbaI recognition site at the 5' end and an EcoRI recognition site at the 3' end were added by PCR using cDNA synthesized by the reverse transcription reaction of RNA prepared from the leaves of each of these six wheat varieties as a template ORF was obtained. For the gene fragment, the TaQ8GTC1 gene treated with XbaI and EcoRI was used as an insert, and pBI121 treated with the same restriction enzyme was used as a vector. These inserts and vectors were combined, and the resulting vector was transformed using the reaction solution, and introduced into Escherichia coli JM109 strain. Then, a plasmid was prepared from a colony in which introduction of the target plasmid was confirmed by colony PCR. Then, a sequencing reaction was performed to analyze the nucleotide sequence of the target gene inserted into the vector.

The primer sets used for PCR are shown below.

TaQ8GTC1-3: 5'-AAAAAATCTAGAGATCTTCAAGAAGCGGAA-3' (sense: SEQ ID NO: 27)

TaQ8GTC1-4: 5'-AAAAAAGAATTCTCACTTCTCTGCCTTCTTTCCGA-3' (antisense: SEQ ID NO: 28)

The primer sets used for sequencing are shown below.

Sequence (TaQ8GTC1)-2: 5'-GACCTCACCATCTTCGAGTC-3' (sense: SEQ ID NO: 29)

Sequence (TaQ8GTC1)-3: 5'-CTCGTACACGTCGAACAG-3' (antisense: SEQ ID NO: 30)

The TaQ8GTC1 gene obtained from the six varieties had the same nucleotide sequence, but was different from the sequence registered in the database. Specifically, the nucleotide sequence determined in this experiment and the nucleotide sequence registered in the database were different in 25 bases out of 675bp constituting the ORF. The difference was accompanied by a 13 amino acid mutation. Hereinafter, the gene of the nucleotide sequence determined in this experiment is described as "TAQ8GTC1-R gene".

The result of comparing the amino acid sequence shown in FIG. 25 with the result of comparing the nucleotide sequence determined in this experiment with the nucleotide sequence registered in the database is shown in FIG. 26 . In Fig. 25, the difference between TaQ8GTC1-R (top) and the nucleotide sequence of TaQ8GTC1 in the database (bottom) is surrounded by boxes. In Fig. 26, 13 different amino acids between TaQ8GTC1-R (top) revealed by sequencing and the amino acid sequence of TaQ8GTC1 in the database (bottom) are boxed. In addition, the differences between the nucleotide sequence determined in this experiment and the nucleotide sequence registered in the database are summarized in Table 8. In Table 8, "-" means that the nucleotide sequences are different, but the amino acid residues are the same.

base sequence difference difference in amino acid sequence T->C, position 25 Tyr (T)→His (H), position 9 C->T, position 61 - C->G, position 138 - C→A, position 151 - G→A, position 207 - A->G, position 246 - T->A, position 287 Met (M)→Lys (K), position 96 C->G, position 315 - C->G, position 348 - C->G, position 382 Gln (Q)→Glu (E), position 128 G->A, position 394 Ala (A)→Thr (T), position 132 T->G, position 429 Asn (N)→Lys (K), position 143 C→G, position 453 - T→C, position 474 - G→C, position 483 Glu (E)→Asp (D), position 161 G→T, position 484 Ala (A)→Ser (S), position 162 G→C, position 486 G→C, position 487 Val (V)→Leu (L), position 163 G→C, position 498 - T→C, position 517 Phe (F)→Leu (L), position 173 A→C, position 525 - A→T, position 560 Glu (E)→Val (V), position 187 C→T, position 581 Ala (A)→Val (V), position 194 T→C, position 595 Phe (F)→Leu (L), position 199 C→G, position 606 Ser (S)→Arg (R), position 202

In addition, the nucleotide sequence determined in this experiment (ie, the nucleotide sequence of the TaQ8GTC1-R gene) and the amino acid sequence encoded by the nucleotide sequence are shown in SEQ ID NOs: 31 and 32, respectively. In addition, the nucleotide sequence of the TaQ8GTC1 gene registered in the database and the amino acid sequence encoded by the nucleotide sequence are shown as SEQ ID NOs: 33 and 34, respectively.

<Construction of E. coli expression form of TaQ8GTC1-R protein (Fig. 27)>

Using cDNA synthesized by reverse transcription reaction of RNA prepared from the foliage of wheat (Agricultural Forestry No. 61) as a template, PCR reaction was performed with TaQ8GTC1-Z and TaQ8GTC1-4 primer sets (see below) to recognize NdeI at the 5' end An ORF of the TaQ8GTC1-R gene with an EcoRI recognition site added to the site, 3' end was obtained. For this fragment, the TaQ8GTC1-R gene treated with NdeI and EcoRI was used as an insert, and pET22b(+) treated with the same restriction enzyme was used as a vector. The insert and the vector were combined, and the vector constructed using the reaction solution was transformed and introduced into Escherichia coli JM109 strain.

Then, a plasmid was prepared from a colony in which introduction of the target plasmid was confirmed by colony PCR. By sequencing, the nucleotide sequence of the TaQ8GTC1-R gene inserted between the NdeI and EcoRI cut sites was checked for errors by PCR, and the plasmid was introduced into E. coli BL21 (DE3) strain E. coli expression form of TaQ8GTC1-R protein. (KTLB-862) was constructed. The primer sets used for PCR are shown below.

TaQ8GTC1-Z: 5'-AAAAAACATATGGCGGCGCCGGCGGTGAAGGTG-3' (sense: SEQ ID NO: 35)

TaQ8GTC1-4: 5'-AAAAAAGAATTCTCACTTCTCTGCCTTCTTTCCGA-3' (antisense: SEQ ID NO: 36)

<Preparation of TaQ8GTC1-R protein>

TaQ8GTC1-R protein was expressed using the constructed E. coli BL21 (DE3) strain (KLB-862). A single colony was inoculated into a liquid LB medium containing 50 ppm of ampicillin, and a solution cultured in a test tube overnight was used as a preculture solution. 2.5 ml of the pre-culture solution was added to 250 mL liquid LB medium (1 L Erlenmeyer flask) containing 50 ppm of ampicillin, and _{cultured at 37° C. and 200 rpm until OD 600} = 0.5-0.6. Then, after cooling on ice for 5 minutes, IPTG was added to a final 1 mM, and TaQ8GTC1-R protein expression was induced at 27°C and 200 rpm for 21 hours, and then collected (4°C, 6000 x g for 10 minutes). centrifugation) and the cells were stored at -80°C. 30mL of PBS buffer (0.14M NaCl, 2.7mM KCl, 10mM Na ₂ HPO ₄ , 1.8mM KH ₂ PO ₄ , pH 7.3) was added to cryopreserved cells (0.5L culture solution), followed by sonication (TAITEC VP-30S micro Chip, output 3, 15 sec constant, 7 to 8 times), and then centrifuged for 20 minutes at 4° C. and 15,000×g to obtain a supernatant. The coenzyme solution was loaded onto the GSTrap FF column and GSTrap 4B column (both bed vol. 1 mL) equilibrated with PBS buffer at a flow rate of 1 mL/min, washed with 20 ml or more of PBS buffer, and both glutathione affinity columns were eluted with 2 mL lysis buffer (50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0) was injected into the column to elute the TaQ8GTC1-R protein (FIG. 28). This protein solution was substituted with PBS buffer by ultrafiltration using Nanosep (10K), and stored at -80°C. Protein concentration was measured by the Bradford method according to the TaKaRa Bradford Protein Assay Kit (Takara) manual.

<Analysis of glutathione conjugation activity of TaQ8GTC1-R protein>

In this experiment, the glutathione conjugation reaction of purified TaQ8GTC1-R protein to CDNB (2,4-dinitrochlorobenzene) and pyroxasulfone was analyzed.

The activity against CDNB was 634 μl of 100 mM potassium phosphate buffer (pH 7.6), 300 μl of 3.3 mM reduced glutathione, 33 μl of purified enzyme (PBS solution containing 20% glycerol), 33 μl of 30 mM CDNB (ethanol solution). It was measured in a total of 1 ml of the reaction solution containing After preparing a reaction solution excluding CDNB, 30 mM CDNB was added at the end to start the enzymatic reaction, and absorbance at 340 nm was measured at 30°C. Activity was calculated by molecular extinction coefficient 9.6mM ^-1 cm ^{-1 .} The reaction solution to which PBS containing 20% glycerol was added instead of the enzyme was used as a negative control (non-enzyme reaction group), and the production amount in this reaction solution was set as the non-enzyme production amount.

The activity against pyroxasulfone was measured in 50 μl of 100 mM potassium phosphate buffer (pH 6.8), 10 μl of 1 mM pyroxasulfone (acetone solution), 20 μl of 10 mM reduced glutathione (pH 7.0), and 120 μl of TaQ8GTC1- R protein (PBS solution containing 20% glycerol) was carried out in a total reaction mixture of 200 μl and reacted at 30° C. for 1 hour, then the reaction was filtered using a 0.2 μm filter, and 50 μl of it was injected into HPLC. did Enzyme-free (PBS containing 20% glycerol was added instead of TaQ8GTC1-R protein) and enzyme and glutathione-free (20% glycerol and PBS instead of TaQ8GTC1-R protein, sterile water was added instead of glutathione) were used as controls. Under the HPLC conditions described below, the retention times of pyroxasulfone and M-15 (glutathione conjugate of pyroxasulfone) were 19.0 minutes and 11.0 minutes, respectively.

Device: Agilent 1100 series

Column: CAPCELL PAK C18 AQ 4.6 mm (I.D.) x 250 mm (SHISEIDO)

Mobile phase: acetonitrile/water = 5/95 (hold for 5 minutes) → (4 minutes) → 40/60 (hold for 4 minutes) → (4 minutes) → 90/10 (hold for 8 minutes) → (1 minute) → 5 /95 (hold for 4 minutes) (each solvent contains 0.5% acetic acid)

Temperature: 35℃

Flow rate: 1.0 mL/min

Detection: 254nm

<Results and considerations>

First, in order to determine whether the purified TaQ8GTC1-R protein in this experiment has GST activity, the glutathione conjugation activity with respect to the standard substrate CDNB (1-chloro-2,4-dinitrobenzene) was investigated. As a result, it was found that the activity against CDNB was 2.17 μmol/min/mg protein (n=2), and the purified enzyme had GST activity.

Next, the conjugation activity of the TaQ8GTC1-R protein to pyroxasulfone was investigated. Conjugation activity test was conducted with enzyme addition (enzymatically and non-enzymatically producing glutathione conjugate), enzyme-free (non-enzymatically producing glutathione conjugate), enzyme and glutathione-free (parent compound). ) was carried out in three reaction solutions. The amount of protein added to the enzyme addition port is based on the fact that when the conjugation activity of about 400 ng of TaQ8GTC0 (see Example 1) was investigated under the same conditions, about 50% of the total 10 nmol of the added herbicide was converted into a glutathione conjugate. It was determined to be 500 ng.

As a result of the conjugation activity test by TaQ8GTC1-R protein, when pyroxasulfone was used as a substrate, the glutathione conjugate produced in the enzyme-added and non-enzyme-added groups was the same (about 3% of the pyroxasulfonic acid added to the reaction solution was M- 15) (Fig. 29). As such, it was found that there was no significant difference in the amount of glutathione conjugate produced in the enzyme-added group and the enzyme-free group, so that the purified TaQ8GTC1-R protein in this test example did not have a conjugation activity to pyroxasulfone. As shown in Example 1 and the like, TaQ8GTC0 having about 80% amino acid homology with TaQ8GTC1-R showed particularly high conjugation activity to pyroxasulfone among VLCFAE inhibitors, whereas TaQ8GTC1-R protein did not.

Since there was no conjugation activity for pyroxasulfone even in GST with relatively high similarity, the pyroxasulfone conjugation activity of TaQ8GTC0 shown in Examples 1 to 3 was considered to have high specificity.

On the other hand, with respect to the amino acid residues involved in substrate recognition of plant GST classified into the Phi class, such as TaQ8GTC0 (TaGSTF3) having pyroxasulfone metabolic activity in wheat-derived GST shown in Examples 1 to 3, the substrate and GST It was revealed by X-ray crystal structural analysis of the complex (P Reinemer et al., J. Mol. Biol. 255, 289-309 (1996); T. Neuefeind et al., J. Mol. Biol. 274, 446-453 (1997)) H. Pegeot et al., Frontiers in Plant Science 5, 1-15 (2014); and T. Neuefeind et al., J. Mol. Biol. 274, 446-453 (1997)). The putative substrate recognition site in the multiple alignment of various plant GST amino acid sequences is shown in FIG. 30, and the amino acids constituting the substrate recognition site revealed in the above paper are summarized in Table 9. In FIG. 30, two putative substrate recognition sites are grouped with a box, and amino acids constituting the putative substrate recognition site are indicated in bold (with underline).

In addition, the amino acid sequence of Arabidopsis-derived AtGSTF2 shown in FIG. 30 is SEQ ID NO: 37, the amino acid sequence of poplar-derived PttGSTF1 is SEQ ID NO: 38, the amino acid sequence of ZmGSTF1 derived from corn is SEQ ID NO: 39, and the amino acid of wheat-derived TaGSTF1 The sequence was shown as SEQ ID NO: 40, the amino acid sequence of wheat-derived TaGSTF4 as SEQ ID NO: 41, the amino acid sequence of wheat-derived TaGSTF5 as SEQ ID NO: 42, and the amino acid sequence of wheat-derived TaGSTF6 as SEQ ID NO: 43, respectively. In addition, in FIG. 30, the amino acid sequence of wheat-derived TaGSTF2-R is shown as SEQ ID NO: 32, and the amino acid sequence of wheat-derived TaGSTF3 is shown as SEQ ID NO: 2, respectively.

Plant GST Amino acids constituting the substrate recognition site AtGSTF2 ¹⁾ I12, L35, S115, F119, F123 AtGSTF2 ²⁾ H8, A10, S11, L35, F119, F123, Y127, Y178 ZmGSTF1 ²⁾ M10, W12, N13, F35, F114, I118 ZmGSTF1 ³⁾ M10, W12, F114, I118, M121, L122, PttGSTF1 ⁴⁾ L12, T14, L37, H119, F123

In Table 9, 1) is Structure 6, 1445-1452 (1998), 2) is J. Mol. Biol. 255, 289-309 (1996), 3) is described in J. Mol. Biol. 274, 446-453 (1997), and 4) are Frontiers in Plant Science 5, 1-15 (2014).

30 and Table 9, the amino acids involved in substrate recognition (corresponding to the amino acid sequence of TaGSTF3 (TaQ8GTC0)) are mainly distributed in region 1 near the 5th to 15th GST and region 2 near the 115th to 125th. can be known Therefore, these two regions were estimated as substrate recognition sites of GST, and homology with the amino acid sequence of TaGSTF3 (TaQ8GTC0) was investigated for each region (region 1 was 10 amino acids, region 2 was 12 amino acids). As a result, the homology between TaGSTF3 (TaQ8GTC0) and TaGSTF1, TaGSTF2-R TaGSTF4, TaGSTF5 and TaGSTF6 in region 1 was 40%, 80%, and 80%, respectively. 40% and 50%. Further, in region 2, the homology between TaGSTF3 (TaQ8GTC0) and TaGSTF1, TaGSTF2-R TaGSTF4, TaGSTF5 and TaGSTF6 was 45%, 64%, and 55%, respectively. 45% and 18%. TaGSTF1, TaGSTF2-R TaGSTF3 (TaQ8GTC0), TaGSTF4, TaGSTF5 and TaGSTF6 are described in I. Cummins et al., Plant Mol. Biol. 52, 591-603 (2003) disclose wheat-derived GST classified into the Phi class.

In both regions 1 and 2, it can be seen that TaGSTF2-R (TaQ8GTC1-R) has the highest amino acid homology to TaQ8GTC0 (TaGSTF3). Considering that TaGSTF2-R (TaQ8GTC1-R) does not exhibit pyroxasulfone metabolic activity, it was considered very specific that TaGSTF3 (TaQ8GTC0) shown in Examples 1 to 3 metabolizes pyroxasulfone.

Comparative Example 3

In this comparative example, the construction of transgenic rice into which the TaQ8GTC1-R gene cloned in Comparative Example 2 was introduced and drug resistance to the transgenic rice were analyzed.

<Construction of TaQ8GTC1-R gene for rice transformation vector (R-5-TaQ8GTC1-R) (FIG. 31)>

A TaQ8GTC1-R gene (ORF) was obtained by performing a PCR reaction with a primer set of TaQ8GTC1-X and TaQ8GTC1-Y using cDNA synthesized by reverse transcription reaction of RNA prepared from the leaves of wheat (Agricultural and Forestry No. 61) as a template. The TaQ8GTC1-R gene fragment treated with SalI at the 5' end and NotI at the 3' end of this fragment was used as an insert, and pENTR-1A treated with the same restriction enzyme was used as a vector. An entry clone (pENTR1A-TaQ8GTC1-R) constructed by combining these inserts and vectors was transformed and introduced into Escherichia coli JM109 strain. Then, a plasmid was prepared from the colony in which the introduction of the target plasmid was confirmed by colony PCR, and it was confirmed by sequencing that the nucleotide sequence of the TaQ8GTC1-R gene inserted between the SalI and NotI cut sites was correct.

Then, the constructed entry clone (pENTR1A-TaQ8GTC1-R) and R-5 (PalSelect pSTARA), the target vector, were subjected to LR reaction and the expression vector constructed (R-5 attB sequence of attB in which the TaQ8GTC1-R gene was inserted) vector for transformation) was introduced into E. coli HST02 strain by transformation. Then, a plasmid was prepared from a colony in which the introduction of the target plasmid was confirmed by colony PCR, and the nucleotide sequence of the TaQ8GTC1-R gene inserted between the attB sequences was confirmed by sequencing analysis. The primer sets used for PCR are shown below.

TaQ8GTC1-X: 5'-AAAAAAGTCGACATGGCGGCGCCGGCGGTGAAGG-3' (sense: SEQ ID NO: 44)

TaQ8GTC1-Y: 5'-AAAAAAGCGGCCGCTCACTTCTCTGCCTTCTT-3' (antisense: SEQ ID NO: 45)

<Introduction of TaQ8GTC1-R gene into rice by transformation>

A rice transformation vector (PalSelect R-5-TaQ8GTC1-R) in which the constructed TaQ8GTC1-R gene was inserted into PalSelect R-5 was introduced into Agrobacterium (EHA105) by electroporation (KLB-872). Then, the TaQ8GTC1-R gene was introduced into the cultured rice cells by the Agrobacterium method, and then selected with bispiribac sodium salt (BS).

TaQ8GTC1-R gene was transformed and introduced into rice, and cultured cells from which the TaQ8GTC1-R gene was introduced were selected with 0.25 μM bispiribac sodium salt (BS) for 1 month, Cultured cells were identified (FIG. 32). After transplanting the cultured cells of this transgenic rice into a drug-free redifferentiation medium, the obtained plants were sequentially cultivated in an isolation greenhouse. Progeny seeds (T1) were plucked from these plants for which ear emergence was confirmed in all plants at an average of 2 months from the start of cultivation in the quarantine greenhouse.

<TaQ8GTC1-R transgenic rice sensitivity test to pyroxasulfone>

For two strains (KLB-872 #3-11 and KLB-872 #2-4), resistance to pyroxasulfone was investigated in a germination inhibition test using gellan gum as a medium. First, hogland mix and 3 g of gellan gum were suspended in 1 L of distilled water, and then thoroughly dissolved in a microwave oven. 15 ml of it was injected into a tubular bottle before cooling (30 ml injected into the plate). The gellan gum medium was injected into the tubular bottle or plate, and the drug was added and mixed well. The rice hull was soaked in a 50-fold diluted sodium hypochlorite solution (antiformin, Wako) for about 20 minutes and then washed well. The sterilized seeds were soaked in distilled water and left at 27°C until germination (about 2 days). The germinated seeds were lightly transplanted into the gellan gum medium (plate) containing 0.25 μM BS with the sprout facing up. These and a beaker containing distilled water were put together in a transparent case, covered with a wrapper, and grown for 2 days at 27° C. under fluorescent lighting (light 14 hours, dark 10 hours). Then, based on the height of the root, the seeds of rice judged to have BS resistance were treated with pyroxasulfone (final concentration 10 ^-8 , 10 ^-7 , 10 ^-6 , 10 ^-5 M) in gellan gum medium ( tubular disease). Then, these and a beaker containing distilled water were put together in a transparent case, covered with a lid, and grown at 27° C. under fluorescent lighting (light light 14 hours, dark light 10 hours) for 4 to 5 days, and then the plant height was measured. Inhibition of the drug was calculated by using the plant height as an index to calculate the growth inhibition rate for the group without the drug, and the 50% growth inhibition concentration (IC ₅₀ value) was measured by the Probit method.

<Results and considerations>

33 shows the results of the above-described TaQ8GTC1-R transgenic rice (KLB-872 #3-11 and KLB-872 #2-4) resistance test to pyroxasulfone. In both strains tested, the sensitivity of pyroxasulfone was the same as that of the wild type. From these results, it was estimated that the TaQ8GTC1-R transgenic rice did not exhibit resistance to pyroxasulfone.

In this example, transgenic rapeseeds (Brassica napus) into which the Q8GTC0 gene, which was found to have a conjugation activity for pyroxasulfone in Examples 1 to 3, was introduced, was prepared, and the sensitivity of the transgenic rapeseed to pyroxasulfone was reduced. tested.

<Construction of TaQ8GTC0 gene for rapeseed transformation vector (FIG. 34)>

PCR reaction was performed with a primer set of TaQ8GTC0 to IF-Blunt End (XbaI) and TaQ8GTC0 to IF-Blunt End (SacI) using cDNA synthesized by reverse transcription reaction of RNA prepared from the leaves of wheat (Agricultural Forestry and Forestry No. 61). was performed to obtain an ORF of the TaQ8GTC0 gene. After treatment with this gene fragment with XbaI and SacI, _{an In-Fusion reaction was performed using pBI121 blunted with T 4} DNA polymerase, and the constructed rapeseed transformation plasmid (TaQ8GTC0 in pBI121) was transformed into E. coli JM109 strain. introduced. Then, a plasmid was prepared from the colony that confirmed the introduction of the target plasmid by colony PCR, and the nucleotide sequence of the TaQ8GTC0 gene inserted between the XbaI and SacI cleavage sites was confirmed by sequencing analysis. Hereinafter, the primers used for PCR were described.

TaQ8GTC0~IF-Blunt End(XbaI):

5'-CACGGGGGACTCTAGATGGCGCCGGCGGTGAAGGT-3' (sense: SEQ ID NO: 46)

TaQ8GTC0~IF-Blunt End(SacI):

5'-GATCGGGGAAATTCGCTACTCTGCTTTCTTTCCAA-3' (antisense: SEQ ID NO: 47)

<Introduction of TaQ8GTC0 gene into rapeseed by transformation>

A vector for rapeseed transformation (pBI121-TaQ8GTC0) in which the constructed TaQ8GTC0 gene was inserted into pBI121 was introduced into Agrobacterium (GV3101) by electroporation (KLB-858). Then, the TaQ8GTC0 gene was introduced into rapeseed by the Agrobacterium method, and then selected with kanamycin.

<Confirmation of genes in transgenic rapeseed>

Genomic DNA of recombinant rapeseed was extracted using DNeasy Plant Mini Kit. Then, introduction of the desired construct was confirmed by PCR using this genomic DNA as a template. PCR was performed in a total of 25 μl of the reaction solution (2 μl template, 0.25 μl of 50 μM sense primer, 0.25 μl of 50 μM antisense primer, 2.5 μl of 2 mM dNTP mixture, 5 μl of Phire Reaction Buffer, 0.5 μl of Phire Hot Start DNA Polymerase 14.5. μl of sterile water). PCR was performed with initial transformation: 98 °C for 20 sec, transformation: 98 °C for 20 sec, ligation: 58 °C for 15 sec, extension: 72 °C for 30 sec (40 cycles), and final extension: 72 °C for 4 min. In addition, the base sequence of the primer used to confirm the introduction of the T-DNA region is as follows.

NOSP-2: 5'-CGCCTAAGGTCACTATCAGCTAGC-3' (antisense: SEQ ID NO: 48)

Linker (pBI121) -2: 5'-GAACTCCAGCATGAGATC-3' (antisense: SEQ ID NO: 49)

CAM35S-1: 5'-AGAGGACCTAACAGAACTCGCC-3' (sense: SEQ ID NO: 50)

TaQ8GTC0-2: 5'-AAAAAACTTAAGCTACTCTGCTTTCTTTCC-3' (antisense: SEQ ID NO: 51)

Next, using the Agrobacterium GV3101 strain (KLB-858) introduced by electroporation with a vector for rapeseed transformation in which TaQ8GTC0 gene was introduced into pBI121 using kanamycin as a selection reagent, the TaQ8GTC0 gene in rapeseed by transformation. was introduced. As a result, one individual considered to be a target transformant was obtained (FIG. 35, the obtained individual was grouped in a box). As a result of PCR reaction using the genomic DNA extracted from this recombinant rapeseed as a template, two structures (PNOS:: NPTII: TNOS, P35S:: TaQ8GTC0:: TNOS) included in the T-DNA region were introduced, and the obtained individual was It was found to be the target recombinant rapeseed (FIG. 36). Regarding this recombinant rapeseed, cultivation was continued in phytotron, and T1 seeds were collected.

<TaQ8GTC0 gene-introduced pyroxasulfone sensitivity test of rapeseed>

[Rapeseed susceptibility test 1 (soil treatment after sowing before germination)]

Rapeseed susceptibility test 1 tested wild-type rapeseed (variety: Westar) and wheat GST transgenic rapeseed. The test scale was a plastic pot (length 8cm, width 8cm, height 6cm), and the test soil was Fujinoto (sandstone), per pot. Four wild-type rapeseed seeds and eight recombinant rapeseed seeds were sown (seed depth of 1 cm). As a drug, a granule hydrate containing 50% pyroxasulfonic acid was tested, and repeated three times at 5 drug doses of 1, 4, 16, 63, and 250 g ai/ha. Immediately after treatment, about 1 mm of rainfall was artificially rained with a rain device, and then water was supplied from the bottom of the port. When the surface of the pot was dried, water was appropriately supplied from the bottom of the pot.

[Results and Discussion]

Soil treatment test (temperature of 22℃, fluorescent lamp) before germination of pyroxasulfone was performed, and the presence or absence of pyroxasulfone resistance of wheat GST recombinant rapeseed rape was analyzed in terms of plant growth and function symptoms and cotyledon growth. Specifically, the plant growth was measured in each treatment group two weeks after sowing of rapeseed seeds and spraying the drug, and the inhibition rate for rapeseed growth was calculated (n=4-12). The test results are shown in FIGS. 37 to 39 . 37 to 39, "TaGST" means TaQ8GTC0 genetically modified rapeseed. The numerical values in the table shown in FIG. 38 mean mean±standard deviation (n=4 to 12).

From the measured inhibition rate on plant growth of wild-type and recombinant rapeseed, the growth inhibition rate of pyroxasulfone for recombinant rapeseed at 4-63 g ai/ha, particularly 16 g ai/ha, was lower than that of wild-type rape (recombinant, 48.2% and 61.9% in the order of wild type). In addition, when the symptom of action was used as an index, the cotyledon of wild-type rape was 16 g a.i./ha or more, whereas this symptom was not confirmed in the case of recombinant rapeseed. Regarding cotyledon growth, wild-type rapeseeds were almost completely suppressed at 16 g a.i./ha or more, whereas recombinant rapeseeds grew correspondingly even at 250 g a.i/ha ( FIG. 39 ).

Summarizing the above results, in the pre-emergence soil treatment test (22℃, fluorescent light) after sowing of pyroxasulfone, rapeseed rapeseed with TaG8QGTC0 gene, which was constructed from the viewpoint of plant growth, action symptoms, and cotyledon growth, was compared with wild type rapeseed. Thus, it was found that it has resistance to pyroxasulfone.

[Rapeseed sensitivity test 2 (agar medium)]

It was found that rapeseed with TaG8QGTC0 gene exhibited resistance to pyroxasulfone in the above-mentioned post-sowing and pre-germination soil treatment test results. Here, in order to find out how many times the resistance of rapeseed to which this TaG8QGTC0 gene has been introduced, the rapeseed growth inhibition test was performed on an agar medium that can directly analyze the effect of the drug. Wild-type rapeseed (cultivar: Westar) and rapeseed with TaQ8GTC0 gene were tested for growth inhibition on agar medium to investigate resistance to pyroxasulfone.

The medium (1 L) in this test is 1X Murashige-Skoog (MS) medium (1 bag), thiamine hydrochloride (3 μg/ml), nicotinic acid (5 μg/ml), pyridoxine hydrochloride (0.5 μg/ ml), 1% (w/v) sucrose. After adding each of the above components to a 1L beaker, the pH was prepared to 5.7, and after filling to 1L, 8g (0.8%) of agar was added. Then, after autoclaving and cooling to room temperature, pyroxasulfone (acetone solution) of a predetermined concentration was added. 50 ml of this solution was dispensed to each plant pot (At this time, a plate was also prepared in which 30 ml of the drug-free solution was dispensed).

When testing the sensitivity of rapeseed, the required amount of dried rapeseed seeds was stirred with 70% ethanol for 2 minutes and hypochlorous acid (0.02% Triton-X-100) for 15 minutes, and washed 10 times with sterile water. About 30 seeds were dented on the surface of the medium of the drug-free plate prepared with these seeds, and after 2-3 days at 22°C, germinated seedlings were buried in each medium (plant box) 5 each. Then, after incubation at 22° C. for 2 weeks, data were obtained.

[Results and Discussion]

The sensitivity test of rapeseed on an agar medium containing pyroxasulfonic acid of various concentrations (0.1 μM, 1 μM, 10 μM, 100 μM) was performed, and the test results are shown in FIGS. 40 to 43 . 40 is a result using wild-type rapeseed, and the numerical values in the table are the mean ± standard deviation (n = 4 to 5). 41 to 43 show the results of using rapeseed (referred to as “TaGST” in the drawing) into which the TaG8QGTC0 gene was introduced. The numerical values shown in the table in FIGS. 41 and 42 are the mean±standard deviation (n=4 to 5). The numerical value shown in FIG. 43 is an average value (n=4-5).

From the measured plant growth inhibition rate and growth inhibition rate of the original leaf, the inhibition rate against wild-type rapeseed in the 0.1 μM addition group (8.9±2.8%, 8.4±1.4% in the order of growth inhibition rate of the original leaf) and the 10 μM addition group Inhibition rates (7.8±5.9%, 1.2±2.7% in the order of plant growth inhibition and true leaf growth inhibition) were equal to wheat GST recombinant rapeseed. Therefore, it was found that rapeseed rape introduced with the TaG8QGTC0 gene had about 100 times the pyroxasulfone resistance compared to the wild type.

From the results of the susceptibility test (soil treatment before germination after sowing, agar medium) of rapeseeds introduced with TaG8QGTC0 gene above, rapeseed with TaG8QGTC0 gene has resistance to pyroxasulfone compared to wild-type rapeseeds, The resistance was estimated to be about 100 times. As such, it was found to have pyroxasulfone metabolic activity in Examples 1 to 3. TaQ8GTC0 was also found to function in rapeseed and impart resistance to pyroxasulfone.

In this example, the resistance to high temperature stress of the transgenic Arabidopsis thaliana (see Example 3) introduced with the TaQ8GTC0 gene was tested.

<High temperature stress tolerance test of Arabidopsis thaliana introduced with TaQ8GTC0 gene>

A medium for examining the high temperature tolerance of transgenic Arabidopsis thaliana introduced with the TaQ8GTC0 gene constructed in Example 3 was constructed as follows: First, Murashige-Skoog (MS) medium (1 bag), thiamine hydro Chloride (3mg), nicotinic acid (5mg), pyridoxine hydrochloride (0.5mg), and sucrose (10g) were added to a 1L beaker, and the pH was prepared to 5.7. After filling to 1L, 8g (0.8%) of agar was added. After autoclaving this, it was cooled to room temperature, and 30 ml was dispensed on a plate.

Using this medium, the high temperature stress tolerance test of the transformed Arabidopsis thaliana constructed in Example 3 was performed as follows. The required amount of dry seeds of Arabidopsis thaliana was stirred with 70% ethanol for 2 minutes and hypochlorous acid (0.02% Triton-X-100) for 15 minutes, respectively, and washed 10 times with sterile water. Then, the seeds were suspended in 1 ml of an autoclaved 0.1% agar solution, and the suspension was placed on the surface of the medium to sow about 30 seeds per plate. It was sealed with a surgical tape and left at 4°C for 2 days. After being transferred to 22°C and grown for 11 days, high-temperature stress treatment (50°C, 40 minutes) was performed, and growth was further performed for 6 days.

[Results and Discussion]

After high-temperature stress treatment with wild-type Arabidopsis thaliana (Columbia-0) as a comparative control, the presence or absence of high-temperature tolerance of Arabidopsis thaliana into which the TaQ8GTC0 gene was introduced was investigated using the plant phenotype as an index. In the test, wild-type and three recombinant Arabidopsis thaliana (1-1-3, 1-1-5, 1-2-12) were used. As a result, the proportion of plants that were chlorosis (completely killed) by high-temperature treatment was 74% (26/35) in the wild type, whereas 1-1-3, 1-1-5, 1-1-3 in the TaQ8GTC0 gene recombinant. The numbers were 0% (0/11), 24% (8/34), 26% (10/38) in the order of 2-12 (number of efflorescences/number of tested individuals in parentheses). From these results, it was found that Arabidopsis thaliana introduced with the TaQ8GTC0 gene was less affected by high temperature treatment than the wild type, and that Arabidopsis thaliana into which the TaQ8GTC0 gene was introduced has resistance to high temperature stress. Therefore, considering that the TaQ8GTC0 gene-transduced rice has high temperature tolerance as shown in Example 4, it can be seen that the TaQ8GTC0 gene functions in a variety of plants from monocots to dicotyledons, and can impart not only herbicide resistance but also high temperature stress resistance. could

In this example, the transgenic rapeseed (Brassica napus) into which the TaQ8GTC0 gene prepared in Example 5 was introduced was tested for high temperature resistance.

<High-temperature stress tolerance test of rapeseed with TaQ8GTC0 gene>

A medium for examining the high temperature tolerance of the transgenic rapeseed with the TaQ8GTC0 gene constructed in Example 5 was prepared as follows. First, Murashige-Skoog (MS) medium (1 bag), thiamine hydrochloride (3 mg), nicotinic acid (5 mg), pyridoxine hydrochloride (0.5 mg), and sucrose (10 g) were added to a 1 L beaker and then , pH was prepared to 5.7, and after filling to 1 L, 8 g (0.8%) of agar was added. After autoclaving this, it was cooled to room temperature, and 70 ml was dispensed into the plant pot.

Using these media, the high-temperature stress tolerance test of the transgenic rapeseed prepared in Example 5 was performed as follows. The required amount of dried rapeseed seeds was stirred in 70% ethanol for 2 minutes and hypochlorous acid (0.02% Triton-X-100) for 15 minutes each, and washed 10 times with sterile water. Then, about 5 seeds were dented on the surface of the medium prepared above. After sealing with Parafilm and growing at 22°C for 4 days, high-temperature stress treatment (1 hour at 50°C or 2 hours and 30 minutes at 50°C) was performed, and the cells were grown for an additional 4 days in an isolation greenhouse.

[Results and Discussion]

After high-temperature stress treatment of wild-type rape (Westar) as a comparative control, it was investigated whether or not high-temperature resistance of rapeseed with TaQ8GTC0 gene. As a result, in Test 1 (treated at 50° C. for 1 hour), the plant length of rapeseeds introduced with the TaQ8GTC0 gene was significantly larger than that of the wild type ( FIG. 44 ). The values in the table shown in FIG. 44 represent the mean±standard deviation (n=3-7). In Test 2 (treated for 2 hours and 30 minutes at 50°C), the percentage of individuals who whitened (completely killed) was 75% (3/4), 20% (1/5) in the order of wild-type rapeseed and TaQ8GTC0 gene introduced. , and the efflorescence ratio of the rapeseed with the TaQ8GTC0 gene was significantly smaller than that of the wild-type rapeseed (the number of efflorescences/tested individuals in parentheses). These results show that, in both tests, using the ratio of planted and whitened individuals as an index, the rapeseed rape introduced with the TaQ8GTC0 gene is less affected by high temperature treatment than the wild-type rapeseed, and the TaQ8GTC0 gene prepared in Example 5 is introduced. was found to have high temperature stress resistance. Therefore, it was found that the wheat-derived TaQ8GTC0 gene also functions in rapeseed, and can confer not only herbicide tolerance but also high temperature stress tolerance.

SEQUENCE LISTING <110> Kumiai Chemical Industry Co., Ltd. <120> A transformed plant having herbicide tolerance <130> PH-6982-PCT <150> JP 2016-132689 <151> 2016-07-04 <160> 51 <170> PatentIn version 3.5 <210> 1 <211> 669 <212> DNA <213> Triticum aestivum <220> <221> CDS <222> (1)..(669) <400> 1 atg gcg ccg gcg gtg aag gtg tac ggg tgg gcc gtg tcg ccg ttc gtg 48 Met Ala Pro Ala Val Lys Val Tyr Gly Trp Ala Val Ser Pro Phe Val 1 5 10 15 gcg cgc cca ctg ctg tgc ctg gag gag gcc ggc gtc gag tac gag ctc 96 Ala Arg Pro Leu Leu Cys Leu Glu Glu Ala Gly Val Glu Tyr Glu Leu 20 25 30 gtg tcc atg agc cgc gcg gcc ggc gac cac cgc cag ccg gac ttc ctc 144 Val Ser Met Ser Arg Ala Ala Gly Asp His Arg Gln Pro Asp Phe Leu 35 40 45 gcc cgg aac ccc ttc ggc cag ctc gag gac ggc gac ctc 192 Ala Arg Asn Pro Phe Gly Gln Val Pro Val Leu Glu Asp Gly Asp Leu 50 55 60 acc ctc ttc gag tcg cgc gcg atc gcg agg cac Glugtg ctc cgg aag cac 240 Thr Leu Phe Ile Ala Arg His Val Leu Arg Lys His 65 70 75 80 aag ccg gag ctg ctg ggc tgc ggc tcg ccg g ag gcg gag gcg atg gtg 288 Lys Pro Glu Leu Leu Gly Cys Gly Ser Pro Glu Ala Glu Ala Met Val 85 90 95 gac gtg tgg ctg gag gtg gag gcc cac cag tac aac Glucc gcg gcc agc 336 Asp Val Glu Ala Met Val 85 90 95 gac gtg Ala His Gln Tyr Asn Pro Ala Ala Ser 100 105 110 gcc atc gtg gtg cag tgc atc atc ttg ccg cta ctg ggc ggc gcg cgg 384 Ala Ile Val Val Gln Cys Ile Ile Leu Pro Leu Leu Gly Gly Ala Arg 115 120 125 gcg gtg gtg gac gag aac gta gcc aag ctc aag aag gtg ctg 432 Asp Gln Ala Val Val Asp Glu Asn Val Ala Lys Leu Lys Lys Val Leu 130 135 140 gag gtg tac gag gca cggac ctg tcg gcg tc gcc ggg gcg tc agg 480 Glu Val Tyr Glu Ala Arg Leu Ser Ala Ser Arg Tyr Leu Ala Gly Asp 145 150 155 160 gac atc agc ctc gcc gac ctc agc cac ttc ccc ttc acg cgc tac ttc 528 Asp Ile Ser Leu Ala Asp Leu Ser Leu Ala Asp Leu Thr Arg Tyr Phe 165 170 175 atg gag acg gag tac gcg ccg ctg gtg gcg gag ctc ccc cac gtg aac 576 Met Glu Thr Glu Tyr Ala Pro Leu Val Ala Glu Leu Pro His Val Asn 180 185 190 gcg tgg tgg gag ggg ctc aag gcc gc agg gtg acg 624 Ala Trp Trp Glu Gly Leu Lys Ala Arg Pro Ala Ala Arg Lys Val Thr 195 200 205 gag ctc atg ccg ccg gac ctt ggg ctt gga aag aaa gca gag tag 669 Glu Leu Met Pro Pro Asp Leu Gly Leu Gly Ala Glu 210 215 220 <210> 2 <211> 222 <212> PRT <213> Triticum aestivum <400> 2 Met Ala Pro Ala Val Lys Val Tyr Gly Trp Ala Val Ser Pro Phe Val 1 5 10 15 Ala Arg Pro Leu Leu Cys Leu Glu Glu Ala Gly Val Glu Tyr Glu Leu 20 25 30 Val Ser Met Ser Arg Ala Ala Gly Asp His Arg Gln Pro Asp Phe Leu 35 40 45 Ala Arg Asn Pro Phe Gly Gln Val Pro Val Leu Glu Asp Gly Asp Leu 50 55 60 Thr Leu Phe Glu Ser Arg Ala Ile Ala Arg His Val Leu Arg Lys His 65 70 75 80 Lys Pro Glu Leu Leu Gly Cys Gly Ser Pro G lu Ala Glu Ala Met Val 85 90 95 Asp Val Trp Leu Glu Val Glu Ala His Gln Tyr Asn Pro Ala Ala Ser 100 105 110 Ala Ile Val Val Gln Cys Ile Ile Leu Pro Leu Leu Gly Gly Gly Ala Arg 115 120 125 Asp Gln Ala Val Val Asp Glu Asn Val Ala Lys Leu Lys Lys Val Leu 130 135 140 Glu Val Tyr Glu Ala Arg Leu Ser Ala Ser Arg Tyr Leu Ala Gly Asp 145 150 155 160 Asp Ile Ser Leu Ala Asp Leu Ser His Phe Pro Phe Thr Arg Tyr Phe 165 170 175 Met Glu Thr Glu Tyr Ala Pro Leu Val Ala Glu Leu Pro His Val Asn 180 185 190 Ala Trp Trp Glu Gly Leu Lys Ala Arg Pro Ala Ala Arg Lys Val Thr 195 200 205 Glu Leu Leu Met Pro Pro Asp Leu Gly Leu Gly Lys Lys Ala Glu 210 215 220 <210> 3 <211> 30 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 3 aaaaaaacata tgg cgccggc ggtgaaggtg 30 <210> 4 <211> 30 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 4 aaaaaaggat ccctactctg ctttctttcc 30 <210> 5 <211> 30 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 5 aaaaaagtcg acatggcgcc ggcggtgaag 30 <210> 6 <211> 32 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 6 aaaaaagcgg ccgcctactc tgctttcttt cc 32 <210> 7 <211> 22 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 7 cacgtgctcc ggaagcacaa gc 22 <210> 8 <211> 20 <212> DNA <213> Artificial <220 > <223> Synthetic DNA <400> 8 ccggcaacag gattcaatct 20 <210> 9 <211> 30 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 9 aaaaaatcta gaaaagtgca gggcaaattc 30 <210> 10 < 211> 30 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 10 aaaaaactta agctatggta tgttcccact 30 <210> 11 <211> 30 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 11 aaaaaatcta gaatcttctt ctccgacgag 30 <210> 12 <211> 30 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 1 2 aaaaaactta agctacttgg cgccaaactt 30 <210> 13 <211> 30 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 13 aaaaaatcta gaatggagcc tatgaaggtg 30 <210> 14 <211> 30 <212> DNA < 213> Artificial <220> <223> Synthetic DNA <400> 14 aaaaaactta agtcatggta ttctcccgct 30 <210> 15 <211> 28 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 15 aaaaaatcta gatcgtttcg aggccgat 28 <210> 16 <211> 30 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 16 aaaaaactta agtcacttgg ccccgaactt 30 <210> 17 <211> 29 <212> DNA <213> Artificial < 220> <223> Synthetic DNA <400> 17 aaaaaatcta gataccagcc acgtcgctt 29 <210> 18 <211> 30 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 18 aaaaaactta agtcacttgg ccccgaactt 30 <210> 19 <211> 28 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 19 aaaaaatcta gagttgggtc tgggacac 28 <210> 20 <211> 30 <212> DNA <213> Artificial <220> <223 > Synthetic DNA <400> 20 aaaaaactta agtcaagcag atggcttcat 30 <210> 21 <211> 30 <212> DNA <213> Artif icial <220> <223> Synthetic DNA <400> 21 aaaaaatcta gaatggccga ggagaagaag 30 <210> 22 <211> 30 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 22 aaaaaactta agctactcga tgcccagcct 30 < 210> 23 <211> 30 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 23 aaaaaagtcg acatggcggc ggcggcggag 30 <210> 24 <211> 33 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 24 aaaaaagcgg ccgctcactt ggccccgaac ttg 33 <210> 25 <211> 30 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 25 aaaaaagtcg acatggcgcc gccgatgaag 30 <210> 26 <211> 33 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 26 aaaaaagcgg ccgcctatgg tatgttcccg ctg 33 <210> 27 <211> 30 <212> DNA <213> Artificial <220> <223 > Synthetic DNA <400> 27 aaaaaatcta gagatcttca agaagcggaa 30 <210> 28 <211> 35 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 28 aaaaaagaat tctcacttct ctgccttctt tccga 35 <210> 29 <211 > 20 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 29 gacctcacca tcttcg agtc 20 <210> 30 <211> 18 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 30 ctcgtacacg tcgaacag 18 <210> 31 <211> 675 <212> DNA <213> Triticum aestivum <220> <221> CDS <222> (1)..(675) <400> 31 atg gcg gcg ccg gcg gtg aag gtg cac ggg tgg gcg atg tcg ccg ttc 48 Met Ala Ala Pro Ala Val Lys Val His Gly Trp Ala Met Ser Pro Phe 1 5 10 15 gtg gcg cgc gcg ttg ctg tgc ctg gag gag gcc ggc gtg gag tac gag 96 Val Ala Arg Ala Leu Leu Cys Leu Glu Glu Ala Gly Val Glu Tyr Glu 20 25 30 ctc ag c cgc gag gcc ggc gac cac cgc cag ccg gac ttc 144 Leu Val Pro Met Ser Arg Glu Ala Gly Asp His Arg Gln Pro Asp Phe 35 40 45 ctc gcc agg aac ccc ttc ggc cag gtc ccc la gtt ctc gag gac gg Arg Asn Pro Phe Gly Gln Val Pro Val Leu Glu Asp Gly Asp 50 55 60 ctc acc atc ttc gaa tcg cgc gcc gtc gcg agg cac gtg ctg cgc aag 240 Leu Thr Ile Phe Glu Ser Arg Ala Val Ala Arg His Val Leu 65 70 75 80 cac aag ccg gag ctg ctg ggc tcc ggc tcg ccg gag tcg gcg gcg aag 288 His L ys Pro Glu Leu Leu Gly Ser Gly Ser Pro Glu Ser Ala Ala Lys 85 90 95 gtg gac gtg tgg ctg gag gtg gag gcg cac cag cac cag acc ccg gcg 336 Val Asp Val Trp Leu Glu Val Glu Ala His Gln His Gln Thr Pro Ala 100 105 110 ggc acc atc gtg atg cag tgc atc ctc acc ccg ttc ctc ggc tgc gag 384 Gly Thr Ile Val Met Gln Cys Ile Leu Thr Pro Phe Leu Gly Cys Glu 115 120 125 cgc gac cag gac gcc g gca aag ctg acg aag ctg 432 Arg Asp Gln Thr Ala Ile Asp Glu Asn Ala Ala Lys Leu Thr Lys Leu 130 135 140 ttc gac gtg tac gag gcg cgg ctg tcg Tyrg tcg agg tac ctc Val Arg gcc ggg Leu Ser Ala Ser Arg Tyr Leu Ala Gly 145 150 155 160 gac tcc ctc agc ctc gcc gac ctc agc cac ttc ccg ctc atg cgc tac 528 Asp Ser Leu Ser Leu Ala Asp Leu Ser His Phe Pro Leu Met 175 175 Tyr 165 atg gac acc gag tac gcg tcg ctg gtg gtg gag cgc ccg cac gtg 576 Phe Met Asp Thr Glu Tyr Ala Ser Leu Val Val Glu Arg Pro His Val 180 185 190 aag gtg tgg tgg gag gag ctc aag gcc agg ccg gcg gcg aag agg gtg 624 Glu Leu Lys Ala Arg Pro Ala Ala Lys Arg Val 195 200 205 acg gag ttc atg ccg cca aac ttc ggg ttc gga aag aag gca gag aag 672 Thr Glu Phe Met Pro Asn Phe Gly Phe Gly Lys Lys Ala Glu Lys tga 675 <210> 32 <211> 224 <212> PRT <213> Triticum aestivum <400> 32 Met Ala Ala Pro Ala Val Lys Val His Gly Trp Ala Met Ser Pro Phe 1 5 10 15 Val Ala Arg Ala Leu Leu Cys Leu Glu Glu Ala Gly Val Glu Tyr Glu 20 25 30 Leu Val Pro Met Ser Arg Glu Ala Gly Asp His Arg Gln Pro Asp Phe 35 40 45 Leu Ala Arg Asn Pro Phe Gly Gln Val Pro Val Leu Glu Asp Gly Asp 50 55 60 Leu Thr Ile Phe Glu Ser Arg Ala Val Ala Arg His Val Leu Arg Lys 65 70 75 80 His Lys Pro Glu Leu Leu Gly Ser Gly Ser Pro Glu Ser Ala Ala Lys 85 90 95 Val Asp Val Trp Leu Glu Val Glu Ala His Gln His Gln Thr Pro Ala 100 105 110 Gly Thr Ile Val Met Gln Cys Ile Leu Thr Pro Phe Leu Gly Cys Glu 115 120 125 Arg Asp Gln Thr Ala Ile Asp Glu Asn Ala Ala Lys Leu Thr Lys Leu 130 135 140 Phe Asp Val Tyr Glu Ala Arg Leu Ser Ala Ser Arg Tyr Leu Ala Gly 145 150 155 160 Asp Ser Leu Ser Leu Ala Asp Leu Ser His Phe Pro Leu Met Arg Tyr 165 170 175 Phe Met Asp Thr Glu Tyr Ala Ser Leu Val Val Glu Arg Pro His Val 180 185 190 Lys Val Trp Trp Glu Glu Leu Lys Ala Arg Pro Ala Ala Lys Arg Val 195 200 205 Thr Glu Phe Met Pro Pro Asn Phe Gly Phe Gly Lys Lys Ala Glu Lys 210 215 220 <210> 33 <211> 675 <212> DNA <213> Triticum aestivum <220> <221> CDS <222> (1)..(675) <400> 33 atg gcg gcg ccg gcg gtg aag gtg tac ggg tgg gcg atg tcg ccg ttc 48 Met Ala Ala Pro Ala Val Lys Val Tyr Gly Trp Ala Met Ser Pro Phe 1 5 10 15 gtg gcg cgc gcg tggc ctg g gcg ggc ctg ctg gag tac gag 96 Val Ala Arg Ala Leu Leu Cys Leu Glu Glu Ala Gly Val Glu Tyr Glu 20 25 30 ctc gtc ccc atg agc cgc gag gcc ggc gac cac cgc cag ccc gac ttc 144 Leu Val Pro Met Ser Arg Glu Ala G His Arg Gln Pro Asp Phe 35 40 45 ctc gcc cgg aac ccc ttc ggc cag gtc ccc gtt ctc gag gac ggc gac 192 Leu Ala Arg Asn Pro Phe Gly Gln Val Pro Val Leu Glu Asp Gly Asp 50 55 60 ctc acc atc ttt tcg cgc gcc gtc gcg agg cac gtg ctg cgc aag 240 Leu Thr Ile Phe Glu Ser Arg Ala Val Ala Arg His Val Leu Arg Lys 65 70 75 80 cac aaa ccg gag ctg ctg gcgc tcc ggc tcg ccg gag at gag tcg g Lys Pro Glu Leu Leu Gly Ser Gly Ser Pro Glu Ser Ala Ala Met 85 90 95 gtg gac gtg tgg ctg gag gtg gag gcc cac cag cac cag acc ccg gcg 336 Val Asp Val Trp Leu Glu Val Glu Ala His Gln His Gln Thr Pro Ala 100 105 110 ggc acc atc gtc atg cag tgc atc ctc acc ccg ttc ctc ggc tgc cag 384 Gly Thr Ile Val Met Gln Cys Ile Leu Thr Pro Phe Leu Gly Cys Gln 115 120 125 cgc gac cag gcc gcc atc gac a ctg acg aat ctg 432 Arg Asp Gln Ala Ala Ile Asp Glu Asn Ala Ala Lys Leu Thr Asn Leu 130 135 140 ttc gac gtg tac gag gcg cgc ctg tcg Asp Tyr Glu A Arg agg tac ctt gcc ggg Ser A Ala Ser Arg Tyr Leu Ala Gly 145 150 155 160 gag gcg gtc agc ctc gcg gac ctc agc cac ttc ccg ttc atg cga tac 528 Glu Ala Val Ser Leu Ala Asp Leu Ser His Phe Pro Phe Met Arg Tyr 165 at 170 175 acc gag tac gcg tcg ctg gtg gag gag cgc ccg cac gtg 576 Phe Met Asp Thr Glu Tyr Ala Ser Leu Val Glu Glu Arg Pro His Val 180 185 190 aag gcg tgg tgg gag gag ttc aag gcc agc g ccg tg 624 Lys Ala Trp Trp Glu Glu Phe Lys Ala Ser Pro Ala Ala Lys Arg Val 195 200 205 acg gag ttc atg ccg cca aac ttc ggg ttc gga aag aag gca gag aag 672 Thr Glu Phe Met Pro Phe Asn Phe Gly Lys Ala Glu Lys 210 215 220 tga 675 <210> 34 <211> 224 <212> PRT <213> Triticum aestivum <400> 34 Met Ala Ala Pro Ala Val Lys Val Tyr Gly Trp Ala Met Ser Pro Phe 1 5 10 15 Val Ala Arg Ala Leu Leu Cys Leu Glu Glu Ala Gly Val Glu Tyr Glu 20 25 30 Leu Val Pro Met Ser Arg Glu Ala Gly Asp His Arg Gln Pro Asp Phe 35 40 45 Leu Ala Arg Asn Pro Phe Gly Gln Val Pro Val Leu Glu Asp Gly Asp 50 55 60 Leu Thr Ile Phe Glu Ser Arg Ala Val Ala Arg His Val Leu Arg Lys 65 70 75 80 His Lys Pro Glu Leu Leu Gly Ser Gly Ser Pro Glu Ser Ala Ala Met 85 90 95 Val Asp Val Trp Leu Glu Val Glu Ala His Gln His Gln Thr Pro Ala 100 105 110 Gly Thr Ile Val Met Gln Cys Ile Leu Thr Pro Phe Leu Gly Cys Gln 115 120 125 Arg Asp Gln Ala Ala Ile Asp Glu Asn Ala Ala Lys Leu Thr Asn Leu 130 135 140 Phe Asp Val Tyr Glu Ala Arg Leu Ser Ala Ser Arg Tyr Leu Ala Gly 145 150 155 160 Glu Ala Val Ser Leu Ala Asp Leu Ser His Phe Pro Phe Met Arg Tyr 165 170 175 Phe Met Asp Thr Glu Tyr Ala Ser Leu Val Glu Glu Arg Pro His Val 180 185 190 Lys Ala Trp Trp Glu Glu Phe Lys Ala Ser Pro Ala Ala Lys Arg Val 195 200 205 Thr Glu Phe Met Pro Pro Asn Phe Gly Phe Gly Lys Lys Ala Glu Lys 210 215 220 <210> 35 <211> 33 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 35 aaaaaacata tggcggcgcc ggcggtgaag gtg 33 <210> 36 <211> 35 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 36 aaaaaagaat tctcacttct ctgccttctt tccga 35 <210> 37 <211> 212 <212> PRT <213> Arabidopsis thaliana < 400> 37 Met Ala Gly Ile Lys Val Phe Gly His Pro Ala Ser Ile Ala Thr Arg 1 5 10 15 Arg Val Leu Ile Ala Leu His Glu Lys Asn Leu Asp Phe Glu Leu Val 20 25 30 His Val Glu Leu Lys Asp Gly Glu His Lys Lys Glu Pro Phe Leu Ser 35 40 45 Arg Asn Pro Phe Gly Gln Val Pro Ala Phe Glu Asp Gly Asp Leu Lys 50 55 60 Leu Phe Glu Ser Arg Ala Ile Thr Gln Tyr Ile Ala His Arg Tyr Glu 65 70 75 80 Asn Gln Gly Thr Asn Leu Leu Gln Thr Asp Ser Lys Asn Ile Ser Gln 85 90 95 Tyr Ala Ile Met Ala Ile Gly Met Gln Val Glu Asp His Gln Phe Asp 100 105 110 Pro Val Ala Ser Lys Leu Ala Phe Glu Gln Ile Phe Lys Ser Ile Tyr 115 120 125 Gly Leu Thr Thr Asp Glu Ala Val Val Ala Glu Glu Glu Ala Lys Leu 130 135 140 Ala Lys Val Leu Asp Val Tyr Glu Ala Arg Leu Lys Glu Phe Lys Tyr 145 150 155 160 Leu Ala Gly Glu Thr Phe Thr Leu Thr Asp Leu His His Ile Pro Ala 165 170 175 Ile Gln Tyr Leu Leu Gly Thr Pro Thr Lys Lys Leu Phe Thr Glu Arg 180 185 190 Pro Arg Val Asn Glu Trp Val Ala Glu Ile Thr Lys Arg Pro Ala Ser 195 200 205 Glu Lys Val Gln 210 <210> 38 <211> 215 <212> PRT <213> Populus trichocarpa <400> 38 Met Ala Thr Pro Val Thr Ile Tyr Gly Pro Pro Leu Ser Thr Ala Val 1 5 10 15 Ser Arg Val Leu Ala Thr Leu Ile Glu Lys Asp Val Pro Phe His Leu 20 25 30 Val Pro Ile Asp Leu Ser Lys Gly Glu Gln Lys Lys Pro Glu Tyr Leu 35 40 45 Lys Ile Gln Pro Phe Gly Gln Val Pro Ala Phe Lys Asp Glu Ser Ile 50 55 60 Thr Leu Phe Glu Ser Arg Ala Ile Cys Arg Tyr Ile Cys Asp Lys Tyr 65 70 75 80 Ala Asp Lys Gly Asn Arg Ser Leu Tyr Gly Thr Asp Ile Leu Ser Lys 85 90 95 Ala Asn Ile Asp Gln Trp Val Glu Thr Asp Gly Gln Thr Phe Gly Pro 100 105 110 Pro Ser Gly Asp Leu Val His Asp Leu Leu Phe Ser Ser Val Pro Val 115 120 125 Asp Glu Ala Leu Ile Lys Lys Asn Val Asp Lys Leu Ala Lys Val Leu 130 135 140 Asp Ile Tyr Glu Gln Lys Leu Gly Gln Thr Arg Phe Leu Ala Gly Asp 145 150 155 160 Glu Phe Ser Phe Ala Asp Leu Ser His Leu Pro Asn Gly Asp Tyr Leu 165 170 175 Val Asn Ser Thr Asp Lys Gly Tyr Leu Phe Thr Ser Arg Lys Asn Val 180 185 190 Asn Arg Trp Trp Thr Glu Ile Ser Asn Arg Glu Ser Trp Lys Lys Val 195 200 205 Leu Glu Met Arg Lys Asn Ala 210 215 <210> 39 <211> 214 <212> PRT <213> Zea mays <400> 39 Met Ala Pro Met Lys Leu Tyr Gly Ala Val Met Ser Trp Asn Leu Thr 1 5 10 15 Arg Cys Ala Thr Ala Leu Glu Glu Ala Gly Ser Asp Tyr Glu Ile Val 20 25 30 Pro Ile Asn Phe Ala Thr Ala Glu His Lys Ser Pro Glu His Leu Val 35 40 45 Arg Asn Pro Phe Gly Gln Val Pro Ala Leu Gln Asp Gly Asp Leu Tyr 50 55 60 Leu Phe Glu Ser Arg Ala Ile Cys Lys Tyr Ala Ala Arg Lys Asn Lys 65 70 75 80 Pro Glu Leu Leu Arg Glu Gly Asn Leu Glu Glu Ala Ala Met Val Asp 85 90 95 Val Trp Ile Glu Val Glu Ala Asn Gln Tyr Thr Ala Ala Leu Asn Pro 100 105 110 Ile Leu Phe Gln Val Leu Ile Ser Pro Met Leu Gly Gly Thr Thr Asp 115 120 125 Gln Lys Val Val Asp Glu Asn Leu Glu Lys Leu Lys Lys Val Leu Glu 130 135 140 Val Tyr Glu Ala Arg Leu Thr Lys Cys Lys Tyr Leu Ala Gly Asp Phe 145 150 155 160 Leu Ser Leu Ala Asp Leu Asn His Val Ser Val Thr Leu Cys Leu Phe 165 170 175 Ala Thr Pro Tyr Ala Ser Val Leu Asp Ala Tyr Pro His Val Lys Ala 180 185 190 Trp Trp Ser Gly Leu Met Glu Arg Pro Ser Val Gln Lys Val Ala Ala 195 200 205 Leu Met Lys Pro Ser Ala 210 <210> 40 <211> 212 <212> PRT <213> Triticum aestivum <400> 40 Met Ala Pro Val Lys Leu Tyr Gly Ala Thr Leu Ser Trp Asn Val Thr 1 5 10 15 Arg Cys Val Ala Ala Leu Glu Glu Ala Gly Val Gln Tyr Glu Ile Val 20 25 30 Pro Ile Asn Phe Gly Thr Gly Glu His Lys Ser Pro Asp His Leu Ala 35 40 45 Arg Asn Pro Phe Gly Gln Val Pro Ala Leu Gln Asp Gly Asp Leu Tyr 50 55 60 Val Phe Glu Ser Arg Ala Ile Cys Lys Tyr Ala Cys Arg Lys Asn Lys 65 70 75 80 Pro Glu Leu Leu Lys Glu Gly Asp Ile Lys Glu Ser Ala Met Val Asp 85 90 95 Val Trp Leu Glu Val Glu Ala His Gln Tyr Thr Ala Ala Leu Ser Pro 100 105 110 Ile Leu Phe Glu Cys Leu Ile His Pro Met Leu Gly Gly Ala Thr Asp 115 120 125 Gln Lys Val Ile Asp Asp Asn Leu Val Lys Ile Lys Asn Val Leu Ala 130 135 140 Val Tyr Glu Ala His Leu Ser Lys Ser Lys Tyr Leu Ala Gly Asp Phe 145 150 155 160 Leu Ser Leu Ala Asp Leu Asn His Val Ser Val Thr Leu Cys Leu Ala 165 170 175 Ala Thr Pro Tyr Ala Ser Leu Phe Asp Ala Tyr Pro His Val Lys Ala 180 185 190 Trp Trp Thr Asp Leu Leu Ala Arg Pro Ser Val Gln Lys Val Ala Ala 195 200 205 Leu Met Lys Pro 210 <210> 41 <211> 222 <212> PRT <213> Triticum aestivum <400> 41 Met Glu Pro Met Lys Val Tyr Gly Trp Ala Val Ser Pro Trp Met Ala 1 5 10 15 Arg Val Leu Val Ser Leu Glu Glu Ala Gly Ala Asp Tyr Glu Leu Val 20 25 30 Pro Met Ser Arg Asn Gly Gly Asp His Arg Arg Pro Glu His Leu Ala 35 40 45 Arg Asn Pro Phe Gly Glu Ile Pro Val Leu Glu Tyr Gly Gly Leu Thr 50 55 60 Leu Tyr Gln Ser Arg Ala Ile Ala Arg His Ile Leu Arg Lys His Lys 65 70 75 80 Pro Gly Leu Leu Gly Ala Gly Ser Leu Glu Glu Ser Ala Met Val Asp 85 90 95 Val Trp Val Asp Val Asp Ala His His Leu Glu Pro Val Leu Lys Pro 100 105 110 Ile Val Trp Asn Cys Ile Ile Asn Pro Phe Val Gly Arg Asp Val Asp 115 120 125 Gly Leu Val Asp Glu Ser Val Glu Lys Leu Lys Lys Leu Leu Glu 130 135 140 Val Tyr Glu Ala Arg Leu Ser Ser Asn Lys Tyr Leu Ala Gly Asp Phe 145 150 155 160 Val Ser Phe Ala Asp Leu Thr His Phe Ser Phe Met Arg Tyr Phe Met 165 170 175 Ala Thr Glu His Ala Val Val Leu Asp Ala Tyr Pro His Val Lys Ala 180 185 190 Trp Trp Lys Ala Leu Leu Ala Arg Pro Ser Val Lys Lys Val Ile Ala 195 200 205 Gly Met Pro Pro Asp Phe Gly Phe Gly Ser Gly Arg Ile Pro 210 215 220 <210> 42 <211> 213 <212> PRT <213> Triticum aestivum <400> 42 Met Ala Pro Ile Lys Leu Tyr Gly Met Met Leu Ser Ala Asn Val Thr 1 5 10 15 Arg Val Thr Thr Leu Leu Asn Glu Leu Gly Leu Glu Phe Asp Phe Val 20 25 30 Asp Val Asp Leu Arg Thr Gly Ala His Lys His Pro Asp Phe Leu Lys 35 40 45 Leu Asn Pro Phe Gly Gln Ile Pro Ala Leu Gln Asp Gly Asp Glu Val 50 55 60 Val Phe Glu Ser Arg Ala Ile Asn Arg Tyr Ile Ala Thr Lys Tyr Gly 65 70 75 80 Ala Ser Leu Leu Pro Thr Pro Ser Ala Lys Leu Glu Ala Trp Leu Glu 85 90 95 Val Glu Ser His His Phe Tyr Pro Pro Ala Arg Thr Leu Val Tyr Glu 100 105 110 Leu Val Ile Lys Pro Met Leu Gly Ala Pro Thr Asp Ala Ala Glu Val 115 120 125 Asp Lys Asn Ala Ala Asp Leu Ala Lys Leu Leu Asp Val Tyr Glu Ala 130 135 140 His Leu Ala Ala Gly Asn Lys Tyr Leu Ala Gly Asp Ala Phe Pro Leu 145 150 155 160 Ala Asp Ala Asn His Met Ser Tyr Leu Phe Met Leu Thr Lys Ser Pro 165 170 175 Lys Ala Asp Leu Val Ala Ser Arg Pro His Val Lys Ala Trp Trp Glu 180 185 190 Glu Ile Ser Ala Arg Pro Ala Trp Ala Lys Thr Val Ala Ser Ile Pro 195 200 205 Leu Pro Pro Ala Val 210 <210> 43 <211> 218 <212> PRT <213> Triticum aestivum <400> 43 Met Ala Pro Val Lys Val Phe Gly Pro Ala Met Ser Thr Asn Val Ala 1 5 10 15 Arg Val Leu Val Cys Leu Glu Glu Val Gly Ala Glu Tyr Glu Val Val 20 25 30 Asp Ile Asp Phe Lys Ala Met Glu His Lys Ser Pro Glu His Leu Val 35 4045 Arg Asn Pro Phe Gly Gln Ile Pro Ala Phe Gln Asp Gly Asp Leu Leu 50 55 60 Leu Phe Glu Ser Arg Ala Ile Ala Arg Tyr Val Leu Arg Lys Tyr Lys 65 70 75 80 Lys Asn Glu Val Asp Leu Leu Arg Glu Gly Asp Leu Lys Glu Ala Ala 85 90 95 Met Val Asp Val Trp Thr Glu Val Asp Ala His Thr Tyr Asn Pro Ala 100 105 110 Ile Ser Pro Ile Val Tyr Glu Cys Ser Ser Thr Ala His Ala Arg Leu 115 120 125 Pro Thr Asn Gln Thr Val Val Asp Glu Ser Leu Glu Lys Leu Lys Asn 130 135 140 Val Leu Glu Val Tyr Glu Ala Arg Leu Ser Lys His Asp Tyr Leu Ala 145 150 155 160 Gly Asp Phe Val Ser Phe Ala Asp Leu Asn His Phe Pro Tyr Thr Phe 165 170 175 Tyr Phe Met Ala Thr Pro His Ala Ala Leu Phe Asp Ser Tyr Pro His 180 185 190 Val Lys Ala Trp Trp Glu Arg Ile Met Ala Arg Pro Ala Val Lys Lys 195 200 205 Leu Ala Ala Gln Met Val Pro Lys Lys Pro 210 215 <210> 44 <211> 34 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 44 aaaaaagtcg acatggcggc gccggcggtg aagg 34 <210> 45 < 211> 32 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 45 aaaaaagcgg ccgctcactt ctctgccttc tt 32 <210> 46 <211> 35 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 46 cacgggggac tctagatggc gccggcggtg aaggt 35 <210> 47 <211> 35 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 47 gatcggggaa attcgctact ctgctttctt tccaa 35 <210> 48 <211 > 24 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 48 cgcctaaggt cactatcagc tagc 24 <210> 49 <211> 18 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 49 gaactccagc atgagatc 18 <210> 50 <211> 22 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 50 agaggaccta acagaactcg cc 22 <210> 51 <211> 30 <212> DNA <213> Artificial <220> <223> Synthetic DNA<400> 51 aaaaaactta agctactctg ctttctttcc 30

Claims (10)

A method for imparting resistance to high temperature stress, comprising introducing a nucleic acid encoding the protein of the following (a) or (b) to a plant having a sensitivity to high temperature stress:
(a) a protein comprising the amino acid sequence of SEQ ID NO: 2;
(b) a protein having an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 2, and having glutathione-S-transferase activity.
According to claim 1,
Plants with sensitivity to high-temperature stress, characterized in that the rice plant
How to give tolerance.
3. The method of claim 2,
Grapeaceae plant, characterized in that rice
How to give tolerance.
The method of claim 1,
Plants with susceptible to heat stress are cruciferous (Brassicaceae), Mesquite (Leguminosae), Apiaceae (Umbelliferae), amaranthaceae (Amaranthaceae), Lamiaceae (Labiatae), chenopodiaceae (Chenopodiaceae), rose family (Rosaceae), Asteraceae (Compositae ), Solanaceae ( Solanaceae ) or Malvaceae ( Malvaceae ) Characterized in that the plant
How to give tolerance.
5. The method of claim 4,
Cruciferous plants are rapeseed ( Brassica napus ) or Arabidopsis thaliana ( Arabidopsis thaliana )
How to give tolerance.
Transgenic plants to which resistance to high temperature stress is imparted by introducing a nucleic acid encoding the protein of (a) or (b) below:
(a) a protein comprising the amino acid sequence of SEQ ID NO: 2;
(b) a protein having an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 2, and having glutathione-S-transferase activity.
7. The method of claim 6,
Plants with sensitivity to high-temperature stress, characterized in that the rice plant
transgenic plants.
8. The method of claim 7,
Grapeaceae plant, characterized in that rice
transgenic plants.
7. The method of claim 6,
Plants with susceptible to heat stress are cruciferous (Brassicaceae), Mesquite (Leguminosae), Apiaceae (Umbelliferae), amaranthaceae (Amaranthaceae), Lamiaceae (Labiatae), chenopodiaceae (Chenopodiaceae), rose family (Rosaceae), Asteraceae (Compositae ), Solanaceae ( Solanaceae ) or Malvaceae ( Malvaceae ) Characterized in that the plant
transgenic plants.
10. The method of claim 9,
The cruciferous plant is a transgenic plant, characterized in that rape ( Brassica napus ) or Arabidopsis thaliana ( Arabidopsis thaliana ).

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